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Title: Influence of hypoxic preconditioning in-vivo to 30 minutes knee surgery specific tourniquet application Name: James Henry Barrington
This is a digitised version of a dissertation submitted to the University of Bedfordshire.
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Influence of Hypoxic Preconditioning in-Vivo to 30 minutes Knee Surgery
Specific Tourniquet Application
By
James Henry Barrington
A thesis submitted to the University of Bedfordshire in partial fulfilment of the
requirements for the degree of Masters of Science by Research
October 2013
I
Abstract
Purpose: To establish whether a bout of hypoxic preconditioning (HPC) or
ischemic preconditioning (IPC) would elicit a reduction in total knee replacement
(TKR) surgery specific tourniquet mediated oxidative stress (OS) in-vivo.
Methods: In an independent group design, 18 healthy men were exposed to 40 min
of either: whole-body HPC (14.3% O2), IPC (four bouts of 5 min ischemia and 5
min reperfusion) or rest (SHAM), 1 h prior to 30 min TKR specific limb ischemia
and 2 h reperfusion. Systemic blood samples were taken at pre- and post-
intervention, additionally blood and gastrocnemius samples were obtained at pre-,
15 min post- (15PoT) and 120 min post-tourniquet deflation. Systemic leukocytes
and gastrocnemius tissue were analysed for the heat shock protein (Hsp72) and
Heat shock protein 32 (Hsp32) gene transcript response (indicates severity of the
cellular stress response), with the systemic plasma also assessed for OS markers
(protein carbonyl and glutathione (reduced, oxidised, total, reduced/oxidised-
ratio)). Results: A 1.93 and 1.97 fold reduction in gastrocnemius Hsp72 was noted
in individuals exposed to HPC (p = 0.007) and IPC (p = 0.006) respectively, in
comparison to SHAM at 15PoT. No significant differences were observed in
gastrocnemius Hsp32, systemic Hsp72, Hsp32 or OS markers (p > 0.05) between
groups. Conclusions: HPC and IPC provided cytoprotection to ischemic stressed
gastrocnemius tissue as indicated by an attenuated cellular stress response to 30
min TKR specific limb ischemia.
II
Author’s Declaration
I declare that the work presented in this thesis is entirely my own.
It has not been submitted for any degree or examination in any other University or
educational institute.
James Barrington
30th
October 2013
III
Table of Contents
Abstract ................................................................................................................ I
Author’s Declaration ........................................................................................... II
List of Figures ................................................................................................... VI
List of Tables .................................................................................................... VII
Acknowledgments ........................................................................................... VIII
Abbreviations .................................................................................................... IX
Chapter 1: Introduction ............................................................................................ 1
1.1 General Introduction ...................................................................................... 2
1.2 Aims and Objectives ..................................................................................... 5
Chapter 2: Literature Review ................................................................................... 6
2.1 Preoperative phase ......................................................................................... 7
2.1.1 Redox Homeostasis................................................................................. 9
2.1.2 Free Radicals........................................................................................... 9
2.1.3 Antioxidant defences ............................................................................ 12
2.1.3.1 Glutathione ..................................................................................... 13
2.1.4 Cellular Damage ................................................................................... 15
2.1.4.1 Lipid Peroxidation .......................................................................... 15
2.1.4.2 Protein Oxidation ........................................................................... 16
2.1.4.3 DNA Damage ................................................................................. 17
2.2 Peri-Operative ............................................................................................. 18
2.2.1 Ischemia ................................................................................................ 18
2.2.2 Reperfusion ........................................................................................... 19
2.2.2.1 Mitochondrial Derived Free Radicals ............................................ 20
2.2.2.2 Xanthine Oxireductase ................................................................... 21
2.2.2.3 Leukocytes ..................................................................................... 23
2.2.3 Glutathione and Ischemia-Reperfusion ................................................ 26
2.2.4 Ischemia-Reperfusion and Macromolecule Damage ............................ 28
2.2.4.1 Apoptotic Cell Death ..................................................................... 29
2.2.4.2 Necrotic Cell Death ........................................................................ 31
2.2.4.3 Toll-Like Receptors ....................................................................... 32
IV
2.3 Ischemic Pre-Conditioning .......................................................................... 34
2.3.1 Early Phase ........................................................................................... 36
2.3.2 Delayed Phase ....................................................................................... 38
2.3.2.1 Antioxidant enzymes ...................................................................... 39
2.3.2.2 Heat shock protein 72..................................................................... 40
2.3.2.3 Heat shock protein 32..................................................................... 44
2.4 Hypoxic Preconditioning ............................................................................. 46
Chapter 3: Methodology ........................................................................................ 49
3.1 Participants .................................................................................................. 50
3.2 Anthropometric Data ................................................................................... 50
3.3 Hydration Status Assessment ...................................................................... 53
3.4 Blood Collection .......................................................................................... 53
3.4.1 K3EDTA Treated Blood........................................................................ 55
3.4.2 Sodium Citrate Treated Blood .............................................................. 55
3.4.3 Lithium Heparin Treated Blood ............................................................ 56
3.5 Muscle Biopsies .......................................................................................... 56
3.6 Experimental Design ................................................................................... 57
3.7 Muscle Sample Preparation ......................................................................... 60
3.8 Protein Carbonyl Quantification.................................................................. 60
3.9 Glutathione Analyses .................................................................................. 62
3.10 RNA extraction .......................................................................................... 64
3.11 RNA concentration quantification ............................................................. 65
3.12 One-step quantitative real-time polymerase chain reaction ...................... 65
3.12 Quantitative real-time polymerase chain reaction analyses ...................... 67
3.13 Statistical Analyses .................................................................................... 68
Chapter 4: Results .................................................................................................. 70
4.1 Circulatory stress and redox markers .......................................................... 72
4.2 Localised muscle stress markers ................................................................. 74
Chapter 5: Discussion ............................................................................................ 76
5.1 Circulatory redox and stress markers .......................................................... 77
5.2 Muscle HSP expression ............................................................................... 82
5.3 Application of results .................................................................................. 88
V
5.4 Limitations ................................................................................................... 89
5.5 Conclusions ................................................................................................. 90
5.6 Recommendation for future research .......................................................... 92
5.6.1 Determination of redox disturbance and cellular structure in muscle
tissue following HPC in a TKR specific tourniquet application ................... 92
5.6.2 The feasibility and clinical relevance of HPC in TKR surgery - A small
clinical trial .................................................................................................... 93
Chapter 6: References ............................................................................................ 95
6.1 References ................................................................................................... 96
Appendices ........................................................................................................... 123
Appendix A ..................................................................................................... 124
Appendix B ...................................................................................................... 125
Appendix C ...................................................................................................... 129
Appendix D ..................................................................................................... 131
Appendix E ...................................................................................................... 133
Appendix F ...................................................................................................... 136
VI
List of Figures
Chapter 2: Literature Review
Figure 2.1: Mechaisms of the superoxide anion. .................................................. 10
Figure 2.2: Glutathione synthesis and metabolism. .............................................. 14
Figure 2.3: Diagram representing the degradation of hypoxanthine. ................... 21
Figure 2.4: Illustration of potential reactive oxygen species production .............. 26
Figure 2.5: Schematic depicting a list of mediators and the proposed time frame
for the protection offered by ischemic preconditioning. ....................................... 36
Figure 2.6: Heat shock response mechanism. ....................................................... 42
Figure 2.7: Regulation of the HSP32 gene............................................................ 45
Chapter 3: Methodology
Figure 3.1: Image of the venepuncture technique. ................................................ 54
Figure 3.2: Images of the muscle biopsy procedure.. ........................................... 57
Figure 3.3: Experimental design. .......................................................................... 60
Chapter 4: Results
Figure 4.1: Mean HR and oxyhaemoglobin saturation during HYP intervention. 71
Figure 4.2: Mean muscle Hsp72 relative gene expression during PrT, 15PoT and
120PoT in all conditions. ...................................................................................... 74
Figure 4.3: Mean muscle Hsp32 relative gene expression during PrT, 15PoT and
120PoT in all conditions. ...................................................................................... 75
Chapter 5: Discussion
Figure 5.1: Schematic of proposed redox experimental design. ........................... 93
Figure 5.2: Experimental design for clinical trial. ................................................ 94
VII
List of Tables
Chapter 3: Methodology
Table 3.1: Participant demographic data ............................................................... 52
Table 3.2: Primer Sequences ................................................................................. 67
Chapter 4: Results
Table 4.1: Systemic circulatory stress and redox markers throughout the
experimental trial .................................................................................................. 73
VIII
Acknowledgments
I would like to thank my supervisors Dr Lee Taylor and Dr Bryna Chrismas. Their
knowledge, expertise and academic guidance throughout this research process has
been invaluable. I would also like to thank James Tuttle for his help and support
with data collection and sample analysis. Additionally, the support given by the
technical staff within the Sport and Exercise Science Laboratories was gratefully
appreciated. I would also like to thank the participants who took part in the study
for their patience and perseverance throughout the gruelling protocol. Also I
would like to thank Mr Oliver Pearce for arranging surgeons to help with the
study around their busy operating schedules. The delicate balancing act required
to successfully achieve this would have undoubtedly caused a headache or two.
Personally, I would like to thank my parents for their love and encouragement
(and delicious food) throughout my life. Without them I would not be the person I
am today. Finally, I would like to thank my girlfriend Fiona Sharpe. Her love,
support and unending belief in my ability has given me the confidence to pursue
my aspirations.
IX
Abbreviations
°C degrees Celsius
µL microlitre
4-HNE 4-hydroxy-nonenal
ADP adenosine diphosphate
AMP adenosine monophosphate
AP1 activator protein 1
ATP adenosine triphosphate
BH4 5,6,7,8-tetrahydrobiopterin
Ca2+
calcium ion
CO carbon monoxide
cm centimetre
DNA deoxyribonucleic acid
DTNB 5,5′-dithio-bis-2-nitrobenzoic acid
EC endothelial cells
eNOS endothelial nitric oxide synthase
ERK extracellular signal-related kinase
ES effect size
ETC electron transport chain
G gravitational force
GAPDH glyceraldehyde 3-phosphate dehydrogenase
GSH reduced glutathione
GSSG oxidised glutathione
H2O2 hydrogen peroxide
HPC hypoxic preconditioning
HR heart rate
hr hour
HSF1 heat shock factor 1
Hsp heat shock protein mRNA
HSP heat shock protein
HXA hypoxanthine
HYP hypoxic preconditioning group
ICAM-1 intracellular adhesion molecule-1
IPC ischemic preconditioning
IRI ischemia reperfusion injury
JNK c-Jun N-terminal kinases
K+ potassium ion
kg kilogram
L litre
LMM linear mixed model
LP lipid peroxidation
X
MAPK mitogen activated protein kinase
MDA malondialdehyde
mg milligram
min minute
miRNA microRNA
mKATP mitochondrial potassium ATP
mL millilitre
mmHG millimetre of mercury
MMP mitochondrial membrane potential
MnSOD manganese superoxide dismutases
mOsmols∙kgH2O-1
miliosmalitymols per kilogram of water
mPTP mitochondrial permeability transition pore
mTOR mammalian target of rapamycin
NADP nicotinamide adenine dinucleotide phosphate
NADPH reduced nicotinamide adenine dinucleotide phosphate
NF-κB nuclear factor-κB
ng nanogram
nm nanometre
NO nitric oxide
NrF2 nuclear factor E2-related factor 2
O2 oxygen molecule
O2•- superoxide
OH•
hydroxyl radical
ONOO- peroxynitrite
PBS phosphate buffered saline
PC protein carbonyl
PKC protein kinase C
RNA ribonucleic acid
RONS reactive oxygen and nitrogen species
ROS reactive oxygen species
RT reverse transcriptase
RT-PCR real-time polymerase chain reaction
s seconds
SHAM sham-operated group
SNAP s-nitroso-n-acteylpenicilleamine
TBARS thiobarbituric acid reactive substances
TGH total glutathione
TKR total knee replacement
TLR toll-like receptors
TNF-α tumor necrosis factor-α
TOR ischemic preconditioning group
XA xanthine
XI
XO xanthine oxidase
XOR xanthine oxireductase
1
Chapter 1: Introduction
2
1.1 General Introduction
Maintenance of homeostasis is paramount in the human body. This delicate
balance is essential for efficient cellular function and survival of the body as a
whole (Tortora and Grabowski, 1996). Stressful situations invoke disruption to the
redox balance via a sharp increase in reactive oxygen species (ROS), diminishing
endogenous antioxidants (such as the glutathione, MnSOD), thus instigating
oxidative stress (Halliwell and Gutteridge, 2007). Although excess ROS induces
oxidative stress and has been correlated with many disease states, low
concentrations of ROS are essential for routine cellular signalling (Ray et al.,
2012).
Tourniquets are widely used during total knee replacement (TKR) surgery to
provide a bloodless field, improving visualisation of crucial structures and
accelerating the surgical procedure (Smith and Hing, 2010; Estebe et al., 2011).
Tourniquets have been used by medical practitioners since ancient Roman times
(Klenerman, 1962), but there had been very little advancement in their design
until the early 20th
century when Harvey Cushing employed a pneumatic
tourniquet system to limit bleeding during a craniotomy procedure (Fletcher and
Healy, 1983). Nevertheless, there are negative features associated with the
application of tourniquets, including, delayed wound healing, vascular injury and
muscular damage (Estebe et al., 2011; Fitzgibbons et al., 2012), inviting
controversy with regards to their use. In fact, delayed wound healing has been
cited to defer patient discharge, incurring greater financial costs to the healthcare
provider (Drew et al., 2007).
3
TKR surgery can last from (mean ± SD) 79.9 ± 12.7 min (Chang et al., 2012) to
145 ± 25 min (Horlocker et al., 2006) during which time a tourniquet is inflated.
The interruption of blood supply mediated by the tourniquet, induces a hypoxic
environment in the distal tissue (Clarke et al., 2001). Long periods of ischemia
inhibit the regeneration of adenosine triphosphate (ATP) through aerobic sources,
placing greater demands on the anaerobic glycolytic pathway (Ostman et al.,
2004). The demand for ATP exceeds its replenishment leading to eventual
adenine nucleotide degradation to produce the purine bases hypoxanthine and
xanthine (Jennings and Reimer, 1991).
Upon reperfusion, the influx of oxygen initiates a rapid production of ROS via
activated leukocytes, enzymatic degradation of purine bases and disruption of the
vital mitochondrial electron transport chain (Granger et al., 1986; Carden and
Granger, 2000; Murphy and Steenbergen, 2008). The large influx of ROS
subsequently overwhelms the endogenous antioxidant defence systems
stimulating oxidative stress (Adachi et al., 2006). Consequentially, triggering
macromolecule damage to enzymatic structures, cellular lipid membranes and
deoxyribonucleic acid (DNA) (Adachi et al., 2006; Ray et al., 2012; Brierley and
Martin, 2013), thus inducing eventual cellular apoptosis and necrosis to the
localised tissue (Jaeschke and Lemasters, 2003). This cascade of events has been
dubbed ischemia reperfusion injury (IRI).
To diminish IRI, many agents and techniques have been applied, including
administration of exogenous antioxidants such as, curcumin (Avci et al., 2012),
caffeic acid (Ozyurt et al., 2006) and edaravone (Hori et al., 2013). Interestingly,
the use of short cycles of ischemia and reperfusion primed the intended tissue and
4
bestowed protection for future ischemic insults (Murry et al., 1986). This
phenomenon was termed ischemic preconditioning (IPC) and has been
demonstrated to provide protection from IRI in both animal models (Mayr et al.,
2004) and human participants (Koca et al., 2011). It is proposed that IPC operates
in a biphasic modus through the activation of protein kinases, heat shock proteins
(HSPs), de novo protein synthesis and stimulated transcription factors (Das and
Das, 2008).
Various animal studies have used hypoxic stress as a preconditioning mediator to
induce similar effects to IPC (Xi et al., 2002; Berger et al., 2010). It has also been
demonstrated that consecutive whole-body hypoxic exposures attenuate the
disruption to the redox balance caused by aerobic exercise in humans (Taylor et
al., 2012). Indeed, hypoxic preconditioning (HPC) has been cited to function
through similar mechanisms to IPC (Zuo et al., 2013). However, very few studies
have investigated the effects of HPC on IRI in human skeletal muscle in vivo.
Since HPC offers protection from redox balance disturbances (Taylor et al., 2012)
and induces similar effects as IPC in animal models (Berger et al., 2010), it is
therefore inferred that HPC would mitigate IRI in human skeletal muscle.
Considering that approximately 153,000 TKR operations were performed in
England and Wales between 2008 and 2010 (Baker et al., 2013) costing on
average £7500 (Dakin et al., 2012), the majority of the financial costs associated
with TKR are incurred following the surgery via patient length of stay (Smith et
al., 2008). Therefore, potential interventions to lessen tourniquet mediated tissue
damage and delayed wound healing are crucial to reducing the length of stay and
thus the financial burden upon health service providers.
5
1.2 Aims and Objectives
This thesis proposes to:
1) Quantify the time course for redox disturbances to the systemic and
localised circulation via analysis of protein carbonyl (PC), reduced
glutathione (GSH), oxidised glutathione (GSSG) and total glutathione
(TGH), following hypoxic and ischemic preconditioning, in addition to
immediately and 2 hrs succeeding tourniquet mediated ischemia.
2) Examine the time course for changes in Hsp72 and Hsp32 in localised
skeletal muscle, in addition to localised and systemic leukocytes utilising
the same time points as outlined in 1).
3) Evaluate the efficacy of both whole-body HPC and limb IPC based on the
observed changes in 1) and 2) from TKR specific tourniquet application.
It was therefore hypothesised that:
IPC and HPC would demonstrate a lower expression of Hsp72 in the
localised muscle tissue (gastrocnemius) in comparison to control following
TKR specific tourniquet application.
Localised muscle Hsp32 would increase in HPC and IPC following
tourniquet ischemia stress when compared to control.
The systemic and localised circulatory redox markers (GSSG, PC) and
stress protein expression (Hsp72 and Hsp32) would increase from the
subsequent bout of TKR tourniquet application in the control condition in
comparison to both HPC and IPC.
6
Chapter 2: Literature Review
7
This thesis is not a technical surgical paper, as such, will not contain novel
operative procedures. The subsequent review will encompass a broad surmise of
the major free radical species and the foremost antioxidant defence system,
glutathione, in relation to the disturbed redox induced by the free radical entities.
In addition, it will portray a logical progression of the events generated by the
counter-intuitive phenomenon, IRI. Finally, the mechanisms responsible for
potential preconditioning techniques will be explored, with particular interest
focused upon the cytoprotective protein family, HSPs.
It should be noted that although this thesis concentrates on the negative effects of
ROS, a large body of evidence is available suggesting the role free radicals play in
hormesis (Nikolaidis et al., 2013) and normal cellular signalling, in both muscle
(Powers et al., 2010a) and blood (Nikolaidis and Jamurtas, 2009).
The heart, kidney, liver and brain have been the most frequently studied within
IRI due to the mortality rates associated with the failure of these organs.
Therefore, the subsequent review has attempted to locate research concerning
skeletal muscle, however research on other tissue has been utilised to demonstrate
the point in question if skeletal muscle data is unobtainable.
2.1 Preoperative phase
TKR is an established treatment to alleviate the pain and discomfort associated
with knee osteoarthritis as well as improving quality of life (Woolhead et al.,
2005). Previous TKR studies have shown a mean length of stay of between 7.6 –
13.4 days (Rissanen et al., 1996; Smith et al., 2008; Jonas et al., 2013), which can
be influenced through a variety of patient (social depravity, age, gender) and
8
hospital (peri-operative analgesia and care, recovery programmes) factors (Jonas
et al., 2013). Reduced length of stay has been shown to be positively associated
with increased patient satisfaction (Husted et al., 2008). To obtain patient insight
with regards to the success of the TKR surgery, the Oxford Knee Score was
developed, as potentially patients’ perception of a satisfied outcome may differ
from that of the surgeon (Dawson et al., 1998). Oxford Knee Score is specific to
TKR surgery, with questions relating to pain and everyday movements rather than
clinical and radiological data (Dawson et al., 1998).
There is vast pressure in the current economic climate to diminish National Health
Service expenditure, particularly via cutting expenses (Dakin et al., 2012).
Recently, research has attempted to establish early recovery programmes
following TKR in an attempt to reduce hospital length of stay, with
implementation demonstrating partial success (Smith et al., 2012). However, TKR
often involves the routine application of a tourniquet to provide a bloodless field
and improve visualisation of structures, thus reducing operative times
(Abdelsalam and Eyres, 1995; Memtsoudis et al., 2010). Consequently, the
occluded blood supply induces a hypoxic environment to the distal tissue (Clarke
et al., 2001), increasing the risk of deleterious effects such as nerve palsy,
metabolic disturbances, muscular injury and IRI (Fitzgibbons et al., 2012). The
combination of these negative effects could perhaps explain the delayed discharge
from hospitals noted in tourniquet versus non-tourniquet studies (Estebe et al.,
2011).
Thus to maintain the advantages of tourniquet use (site structure clarity, reduced
operative time) but attempt to minimise the associated deleterious effects, a
9
preconditioning intervention could be utilised. Therefore, it is necessary to
establish homeostatic molecular events in order to comprehend the contrasting
circumstances that occur through tourniquet mediated ischemia.
2.1.1 Redox Homeostasis
Prior to surgery in healthy individuals, redox homeostasis is maintained via an
array of endogenous and exogenous antioxidant defence systems combating
excessive pro-oxidant RONS (reactive oxygen and nitrogen species) (Halliwell
and Gutteridge, 2007). At rest, the continuous production of RONS are associated
with normal cellular metabolism (Valko et al., 2007). However, an increased
production of pro-oxidants and concomitant failure of the anti-oxidant defence
system initiates oxidative stress (Halliwell and Gutteridge, 2007). In this thesis,
oxidative stress will be defined as; a disturbance to the redox balance in favour of
ROS, leading to potential damage (Halliwell and Whiteman, 2004).
Below is a concise description of the major ROS produced by skeletal muscle
tissue, the source of these radicals and their associated interactions with cellular
components.
2.1.2 Free Radicals
Superoxide Anion
Superoxide (O2•-) is produced through the transfer of an electron to the base-state
oxygen atom, generally occurring in the “leaky” sites of the mitochondria electron
10
transport chain or from enzymatic reactions within the cell (Powers et al., 2010a).
O2•- is considered a fairly weak ROS in comparison to others mentioned
subsequently (Halliwell and Gutteridge, 2007), however, although the radical
itself may not react directly, it can induce more potent species through its
conversion (Figure 2.1) and has a relatively long half-life in comparison to other
species (Halliwell, 1999; Powers and Jackson, 2008). Of note is the dismutation
of O2•-
through enzymatic (manganese superoxide dismutases) and spontaneous
reactions providing a key source of hydrogen peroxide (H2O2) (Powers and
Jackson, 2008).
Figure 2.1: Mechanisms of the superoxide anion in initiating damage through its
conversion. Abbreviations: O2•- - superoxide; H2O2 - hydrogen peroxide; OH
• -
hydroxyl radical; HO2• - protonated superoxide anion; NO
• - nitric oxide; ONOO
-
- peroxynitrite; HP - heme proteins; Cp - caeruplasmin. Diagram taken from
Halliwell (1999).
Hydroxyl Radical
11
Hydroxyl radicals (OH•) are highly reactive and due to this, are not membrane
permeable, often reacting immediately with the surrounding environment. The
majority of OH•
formed are transpired through Fenton chemistry of H2O2
catalysed by free heme (Halliwell, 1995). Indeed, research has demonstrated that
the majority of the damage to DNA in cells treated with H2O2 is caused by OH•
(Spencer et al., 1995).
Hydrogen Peroxide
H2O2 is a relatively weak stable oxidising agent, however, it can permeate cell
membranes and is readily produced, not just through dismutation of O2•-
(Powers
and Jackson, 2008) as mentioned previously, but also via other enzymatic
reactions including xanthine oxidase (XO) (Sachdev and Davies, 2008). Although
not very reactive, H2O2 is capable of inactivating certain enzymes directly,
including the glycolytic enzyme GAPDH (Glyceraldehyde 3-phosphate
dehydrogenase) (Halliwell and Gutteridge, 2007) and is the main source of OH• as
mentioned previously.
Nitric Oxide (NO)
Nitric oxide (NO) is soluble in both water and lipids and has an array of biological
roles including, vasodilation, neurotransmission and inhibition of platelet
aggregation (Love, 1999). It is mainly synthesised through the conversion of L-
arginine catalysed by the enzyme nitric oxide synthase (Powers and Jackson,
2008). Although not a reactive free radical, NO reacts readily with O2•- to form the
reactive nitrogen specie, peroxynitrite (ONOO-), which can lead to the production
12
of other damaging species (Brown and Borutaite, 2002). NO will react
preferentially with other radicals and heme groups (Ferrer-Sueta and Radi, 2009).
Peroxynitrite
ONOO- is biologically generated through the reaction between O2
•- and NO
(Ferrer-Sueta and Radi, 2009). Although ONOO- is fairly stable, it is a strong
oxidant, which reacts slowly with biological molecules, therefore largely
influencing biological reactions within cells (Pacher et al., 2007). Upon pronation,
ONOO- is converted to peroxynitrous acid which is extremely reactive and yields
further oxidising and nitrating species (Arteel et al., 1999).
The production of RONS are imperative to correct regulation of wound healing
(Sen, 2003). However, an accumulation of excess RONS leads to oxidative stress
and eventual impairment in wound healing (Soneja et al., 2005). Therefore, it is
inferred that oxidative stress produced via tourniquet inflation during TKR
operations would negatively affect wound healing following surgery. Fortunately,
the body has the ability to negate excessive pro-oxidants and maintain redox
homeostasis via an antioxidant defence system.
2.1.3 Antioxidant defences
The body has a multitude of antioxidant defence systems to defend against
cellular free radical disruption. Endogenous systems include the enzymes
glutathione reductase, catalase and superoxide dismutase among others
(Halliwell, 1999). In addition, exogenous sources that are gained through dietary
intake such as vitamin C and E also supplement the antioxidant defence
13
(Halliwell, 1999). However, detail descriptions of these are not in the scope of this
thesis. Accordingly, following the account above, the subsequent section will
provide a thorough description of the antioxidant system glutathione as this
particular marker was assessed during the experimental trial.
2.1.3.1 Glutathione
Glutathione is the most abundant non-protein thiol found in animal cells which is
synthesised exclusively in the cytosol and is distributed throughout intracellular
organelles including the nucleus, mitochondria and the endoplasmic reticulum
(Mari et al., 2013). Intracellular glutathione is predominantly found as GSH,
accounting for around 99% of the TGH found in the majority of human tissue
(Halliwell and Gutteridge, 2007). Degradation of GSH to its oxidised form
(GSSG) occurs exclusively in extracellular spaces, thus involving membrane
transporters to deliver glutathione to different organelles or extracellular spaces
(Ballatori et al., 2009). The biosynthesis of glutathione involves glutamate,
cysteine and glycine through the enzymatic action of glutamate cysteine ligase
and glutathione synthase (Figure 2.2) (Maher, 2005). GSH can neutralise H2O2
either through enzymatic (glutathione peroxidase) or non-enzymatic reactions
generating GSSG, which is then available to be recycled back into GSH to aid
further in cellular protection (Figure 2.2) (Maher, 2005; Halliwell and Gutteridge,
2007).
14
Figure 2.2: Glutathione synthesis and metabolism pathways. Abbreviations: GSH
– reduced glutathione; GSSG - oxidised glutathione; NADPH – reduced
nicotinamide adenine dinucleotide phosphate; NADP – nicotinamide adenine
dinucleotide phosphate; H2O2 – hypdrogen peroxide; H2O – water. Adapted from
Maher (2005).
Glutathione is often measured in whole blood samples due to the low
concentration of TGH found in plasma (0.5%) and vast levels in erythrocytes
(99.5%) (Serru et al., 2001). It has been previously used in varying protocols to
assess oxidative stress in hypoxia (Taylor et al., 2012), dehydration (Hillman et
al., 2011) and ageing (Jones et al., 2002) among others.
Glutamate + Cysteine
Glutamate Cysteine Ligase
γ-glutamyl-cysteine
Glutathione Synthase
GSH
GSSG NADP
NADPH H2O
H2O2
Glycine
Glutathione Peroxidase Glutathione Reductase
15
The attenuation of GSH has been associated with delayed wound healing (Rasik
and Shukla, 2000), therefore, monitoring concentrations of this tripeptide would
provide useful when assessing tourniquet mediated redox disturbances.
Multiple methods are available to analyse glutathione samples from human blood
and muscle tissue (Rahman et al., 2006), the choice made regarding the presented
thesis is detailed in section 3.9.
2.1.4 Cellular Damage
If the antioxidant defence systems are overwhelmed oxidative stress will ensue
(Halliwell and Gutteridge, 2007). Excessive cellular damage of lipids, proteins
and DNA following oxidative stress is common in delayed wound healing (Soneja
et al., 2005). Below is a general description of the mechanisms that produce lipid
peroxidation, protein oxidation and DNA damage following oxidative stress.
2.1.4.1 Lipid Peroxidation
Free radicals induce structural changes to cellular membranes, affecting their
functional capacity, thus allowing more direct free radical attacks upon
intracellular proteins (Halliwell and Chirico, 1993). In addition, lipid peroxidation
(LP) increases membrane permeability to ions, giving rise to intracellular
increases of Ca2+
inducing disruptions to cellular metabolism (Halliwell and
Chirico, 1993; Niki, 2008).
Radical mediated peroxidation mechanisms involve the removal of protons from
polyunsaturated fatty acids to produce a lipid radical (Gueraud et al., 2010). This
initiates a propagation reaction via lipid radical oxidation, which in turn reacts
16
with a fresh lipid, invariably creating another lipid radical and the unstable lipid
hydroperoxyde, inevitably continuing the chain reaction (Gardner, 1989; Gueraud
et al., 2010). Termination of the reaction only occurs when the creation of non-
radical and non-propagating species transpires (Gueraud et al., 2010).
LP is thought to be associated with various human disease states such as
Alzheimer’s disease (Pratico and Sung, 2004), atherosclerosis (Minuz et al., 2006)
and IRI (Adachi et al., 2006). Measuring LP is usually performed analysing the
stable products of lipid hydroperoxyde, such as malondialdehyde (MDA), 4-
hydroxy-nonenal (4-HNE) or isoprostanes (Powers et al., 2010b). MDA for
example is commonly assessed via the use of thiobarbituric acid reactive species
(TBARS) assay kits, however, most of the TBARS found in human samples
appear not to be related to LP or MDA, therefore use of high performance liquid
chromatography is recommended as a preferred method (Halliwell and Whiteman,
2004).
2.1.4.2 Protein Oxidation
ROS undergo numerous reactions (electron transfer, hydrogen abstraction, re-
arrangement) with protein peptides during oxidative stress, ultimately disrupting
the functionality of the protein structure (Hawkins and Davies, 2001). The
majority of free radical attacks are focused upon the peptide side-chains and the
protein back-bone, forming a multitude of radicals due to the variability of
potential sites upon both of these protein fragments (Hawkins and Davies, 2001).
The predominant product of protein oxidation are carbon-centred radicals, which
are well known precursors to PCs (Sibrian-Vazquez et al., 2010). The most
17
frequently used and reliable technique for assessing protein oxidation is via the
reaction between 2,4-dinitrophenylhydrazine and PC for spectophotometry
analysis, based on methods by Levine et al. (1994). Indeed, PC quantification is
particularly pertinent within this thesis as protein oxidation stimulates the
activation of HSPs and the heat shock response (Noble et al., 2008) (detailed
further in section 2.3.2.1).
2.1.4.3 DNA Damage
DNA damage is considered the most serious of ROS induced alterations as DNA
is merely copied, provoking mutations into the base sequence of replicated
nucleic acids (Poulsen, 2005). The majority of DNA damage is caused by ROS
and it is estimated that 2 x 104 damaging events occur in every human cell every
day (Barzilai and Yamamoto, 2004). This ROS mediated damage occurs due to
oxidising of nucleic bases, splitting DNA cross-links and breaking single/double
DNA strands (Barnes and Lindahl, 2004). Fortunately, the body operates effective
systems in repairing DNA modifications through base-excision and nucleotide-
excision repair pathways (Brierley and Martin, 2013). DNA alteration is
considered to be a pathophysiological factor in the development of cancer
(Poulsen, 2005) and is often detected via oxidised by-products utilising high
performance liquid chromatography and mass spectrometry techniques (Weimann
et al., 2001).
Increased protein damage is associated with delayed wound healing and is
speculated, in part, to be produced via oxidative stress (Moseley et al., 2004).
18
Therefore, alleviation of tourniquet mediated oxidative stress following TKR
surgery may help to reduce wound healing durations.
2.2 Peri-Operative
The induction of oxidative stress through increased radical production and the
concomitant failure in antioxidant defences invariably leads to cellular structural
degradation. The subsequent description will outline the molecular events that
occur during tourniquet inflation in the peri-operative phase of TKR surgery,
eventually leading to the developing stages of oxidative stress and associated
cellular damage.
2.2.1 Ischemia
Tissue distal to the tourniquet becomes ischemic and hypoxic (Clarke et al.,
2001), consequentially disturbing aerobic metabolism, invariably placing greater
demands upon anaerobic sources (Grace, 1994). Oxygen is an essential fuel
source for cellular metabolism, replenishing ATP concentrations via the electron
transport chain (ETC) in the mitochondria (Tortora and Grabowski, 1996).
However, during ischemia, glycolysis becomes the main source of ATP re-
synthesis, but this process in turn, also increases the ratio of nicotinamide adenine
dinucleotide phosphate (NADP)/reduced NADP (NADPH) in addition to lowering
the pH, consequentially slowing the glycolytic process via inhibition of GAPDH
(Jennings and Reimer, 1991). As ATP re-synthesis slows beyond cellular usage,
adenosine diphosphate (ADP) concentrations rise, allowing two of these
molecules to be converted into ATP and adenosine monophosphate (AMP), where
the latter undergoes eventual degradation to hypoxanthine (HXA) if O2 is not
19
restored (Jennings and Reimer, 1991). Under normal homeostatic situations,
metabolites would be removed by the circulation, but during blood flow occlusion
concentrations are allowed to accumulate (Grace, 1994). In addition, the hypoxic
conditions also up-regulate the enzyme xanthine oxidoreductase (XOR) which
catalyses HXA into xanthine (XA) and XA into uric acid in the presence of
oxygen (Hassoun et al., 1998). Limb ischemia can last for up to (mean ± SD) 145
± 25 min during TKR surgery (Horlocker et al., 2006). This prolonged blood
occlusion can lead to significant cell necrosis and tissue damage, therefore
restoration of blood flow is paramount (Lefer and Lefer, 1996).
Paradoxically, ischemia itself appears not to induce as severe damage in
comparison to the re-introduction of oxygen after a bout of blood flow-occlusion
(Parks and Granger, 1986a). This illogical phenomenon and subsequent tissue
damage has been named IRI and has a very complex pathophysiology (Lefer and
Lefer, 1996).
2.2.2 Reperfusion
An appropriate beginning is with the many cellular regions in which oxidative
stress can be derived. Once surgery is completed and the tourniquet has been
deflated, the re-occurrence of blood flow to the hypoxic tissue leads to a rapid
induction of free radicals which has been cited as a major factor of oxidative
stress leading to IRI (Grace, 1994). Outlined below are the major sources of free
radicals which lead to the disruption of the redox balance and consequential
cellular damage, and eventual delay in wound healing following IRI.
20
2.2.2.1 Mitochondrial Derived Free Radicals
The ischemic environment is hypothesised to disrupt the mitochondrial ETC; this
disruption leads to inefficient electron transfer and increased ROS production
upon reperfusion (Murphy and Steenbergen, 2008). Indeed, inhibition of the ETC
at complex I during ischemia has been demonstrated to diminish IRI in cardiac
tissue (Lesnefsky et al., 2004). Three extensively studied factors regulate
mitochondrial ROS production; mitochondrial membrane potential (MMP), Ca2+
concentrations and NO availability (Zhang and Gutterman, 2007). High MMP
promotes ROS generation via slowing of the ETC and prolonging the
ubisemiquinone radical immediate at complex III, known to be a considerable site
of O2•- production (Zhang and Gutterman, 2007).
Mitochondria play a key role in Ca2+
homeostasis, providing a transient location
for Ca2+
storage, elevating inter-mitochondrial membrane Ca2+
concentrations
(Bernardi, 1999). Ca2+
stimulates ROS generation through activation of the
tricarboxylic acid cycle, thus enhancing the work rate of the mitochondrion and
augmenting ROS output (Brookes et al., 2004). Ca2+
can also initiate NO
production, leading to inhibition of complex IV, again enhancing the radical
stimulating intermediate, ubisemiquinone (Cleeter et al., 1994). The ineffective
electron transfer stimulates the production of the free radical O2•-, damaging
mitochondrial proteins and further exacerbating the disruption upon the ETC
(Baines, 2009). The polarity of O2•- makes it difficult for diffusion across the
mitochondria membrane, with the mitochondrial permeability transition pore
(mPTP) serving as the only conduit for its transport (Han et al., 2003). The most
21
important mechanism for intermembrane transport of O2•- is via its conversion
into the uncharged H2O2, which can easily diffuse across membranes into the
cytosol (Zhang and Gutterman, 2007).
The role of mitochondrial derived ROS is well established in the pathophysiology
of sustained ischemia and reperfusion (Lesnefsky et al., 2004; Baines, 2009), thus
would contribute to macromolecule damage and redox disturbances observed in
tourniquet application (Koca et al., 2011).
2.2.2.2 Xanthine Oxireductase
The accumulation of HXA during the ischemic bout allows for its conversion via
XOR once molecular oxygen is restored and has been implemented as a key
contributor of ROS production during ischemia-reperfusion (Granger et al., 1986).
XOR has two inter-convertible forms; XO and xanthine dehydrogenase both of
which are capable of converting HXA and XA, only differing due to the former
able to catalyse exclusively via reducing oxygen (Figure 2.3) (Berry and Hare,
2004).
Figure 2.3: Diagram representing the degradation of hypoxanthine following the
re-introduction of oxygen. Abbreviations: O2 – oxygen molecule; H2O2 –
hydrogen peroxide. Adapted from Berry & Hare (2004).
Hypoxanthine
Xanthine
O2
Xanthine Oxidase
H2O2
Xanthine Oxidase
O2 H2O2
Uric Acid
22
The conversion of XOR to XO is initiated in hypoxic conditions (George and
Struthers, 2009) and is the main component for XOR derived ROS via generation
of by-products H2O2 and O2•- (Berry and Hare, 2004). In mammals, the highest
proportion of XOR is observed in the small intestine and the liver (Parks and
Granger, 1986b), although in humans the majority of XOR is an inactive state
(Berry and Hare, 2004). It has also been reported that it exists solely in the
cystoplasm of cells (Ichikawa et al., 1992), although this is under some debate
(Frederiks and Vreeling-Sindelarova, 2002).
Several lines of evidence have implicated XO in the pathophysiology of IRI.
Anoxic aortic endothelial cells demonstrated large bursts of ROS after ischemia-
reperfusion, and were linked to the activity of XO (Zweier et al., 1994). Indeed,
XO derived by-products have also been linked to cardiac dysfunction in rat hearts
after ischemia-reperfusion, which was reduced following treatment with the ROS
scavenger allopurinol (Brown et al., 1988). Furthermore, implementation of XO
inhibitors abolished free radical creation in rat brain during a bout of ischemia-
reperfusion (Phillis et al., 1994). These findings were also supported by another
XO inhibitor (BOF-4272) which abolished lipid membrane peroxidisation in
hepatocytes in an in vitro model (Kakita et al., 2002). However, there is
controversy to whether XO actually contributes to the ischemia reperfusion
radical production. Lindsay et al. (1990) performed 5 hrs of ischemia upon ex-vivo
canine gracilis muscle and measured small increases in IRI precursors HXA and
XA, in addition to a minor increase in XO activity between 5 and 15 min after
initial reperfusion. Nevertheless, there were no apparent rises in uric acid which
would be the expected by-product from this enzymatic reaction. Surprisingly, this
23
article did fail to assess any markers of tissue damage which may have provided a
clear link to whether the low conversion of HXA/XA to uric acid corresponded to
similar levels of tissue injury.
As tourniquet inflation has been attributed to inducing IRI (Estebe et al., 2011)
and XO is considered to contribute to IRI (Carden and Granger, 2000), it therefore
could be speculated that XO participates in tourniquet mediated ROS generation.
2.2.2.3 Leukocytes
The significance of XO derived ROS in IRI has been disputed with some authors
favouring the role of leukocytes as the major source of ischemia-reperfusion
mediated oxidative stress. Under normal circumstances, synthesis of NO is
produced by endothelial nitric oxide synthase (eNOS) utilising molecular oxygen
and L-arginine in the presence of the essential co-factor 5,6,7,8-
tetrahydrobiopterin (BH4) (Perkins et al., 2012). However, BH4 is easily oxidised
to dihydrobiopterin if oxidant radicals overwhelm key intracellular antioxidant
defence mechanisms such as glutathione or vitamin E (Crabtree et al., 2008). The
reduction in BH4 concentrations cause eNOS to switch from NO production to the
O2•- radical, thus furthering oxidation of BH4 (Schmidt and Alp, 2007; Crabtree et
al., 2008). A reduction in NO release has been cited frequently in relation to IRI
(Lefer and Lefer, 1996; Carden and Granger, 2000; Khanna et al., 2005; Perkins
et al., 2012), and appears to be related to the abrupt release of O2•- upon
reperfusion inhibiting NO synthesis (Ma et al., 1993). Moreover, as mentioned in
section 2.1.2, the increased availability of O2•- provides NO the ability to react and
24
produce the potent radical ONOO- accumulating to the exacerbation of oxidative
stress (Crabtree et al., 2008).
Leukocytes are recruited to the ischemic site via cytokines, chemokines and
cellular selectins expressed on the endothelial cells (ECs) (Jaeschke, 2003).
Leukocytes free-flowing in the microcirculation pass the ECs and respond to
inflammation mediated selectins, progressively slowing in a process known as
leukocyte rolling (Kansas, 1996). P-selectin expressed on ECs has been
implicated as the dominate receptor for leukocyte rolling (Carden and Granger,
2000), which is found within intracellular Weibel-Palade bodies and is rapidly
exteriorised to the cell surface membrane within minutes of agonist (histamine,
thrombin, H2O2) stimulation (Kansas, 1996; Lefer and Lefer, 1996). P-selectin
glycoprotein ligand-1 on the surface of leukocytes is the principle ligand for P-
selectin and results in intergrin activation which links leukocyte rolling with fixed
cellular adhesion (Langer and Chavakis, 2009). ß2 intergrin (CD11b/CD18)
present in leukocytes have also been implemented as the principle molecule for
firm adhesion of leukocytes to the EC membrane via the membrane’s counter-
receptor, intracellular adhesion molecule-1 (ICAM-1) (Lefer and Lefer, 1996).
Once bound, the activated leukocytes release proteases which are capable of EC
membrane degradation, in addition to release of H2O2 and O2•-
inducing oxidative
stress (Carden and Granger, 2000). Furthermore, bound leukocytes infiltrate the
tissue through the endothelial barrier resulting in greater endothelial damage and
edema formation (Nedrebo et al., 2003).
25
Strong evidence implicates the role of leukocytes in IRI. Investigation into mice
deficient in CD11/CD18, P-selectin or ICAM-1 demonstrated complete protection
from mesenteric artery EC injury after a 45 min bout of ischemia-reperfusion
(Banda et al., 1997). These results were corroborated by Connolly et al. (1996)
who found a 3.7 fold reduction in infarct size from 45 min ischemia followed by
22 hrs reperfusion on the cerebral artery in ICAM-1(-/-)
mice compared with
ICAM-1(+/+)
control mice. Furthermore, canines administered with a CD18
intergrin neutralising antibody demonstrated a diminution of myocardial infract
size in comparison to control after ischemia-reperfusion (Duilio et al., 2001). As
previously mentioned, NO has been intrinsically linked with IRI. Indeed,
inhibition of endogenous NO synthesis increased vascular leukocyte rolling in
humans (Hossain et al., 2012). In addition, administration of NO (via NO donor
S-nitroso-N-acteylpenicilleamine (SNAP)) has also shown to be effective in
diminishing leukocyte rolling and adhesion (Dal Secco et al., 2006). However, the
administration of SNAP has been shown to aggravate IRI through greater free
radical generation (Zhang et al., 2003) demonstrating the difficulty of exogenous
NO donor administration in attempting to prevent IRI.
These results clearly depict an integral role for leukocyte, mitochondrion and
XOR in contributing to greater free radical production (Figure 2.4) and ensuing
oxidative stress experienced during IRI. Hence, the oxidants produced through
XOR during initial re-oxygenation suppress NO release and augmented O2•-
production initiating leukocyte rolling and increased oxidant production
respectively, thus inevitably disrupting the redox balance. As redox homeostasis is
paramount in avoiding cellular injury (Zweier and Talukder, 2006), diminishing
26
the associated redox balance disturbances during tourniquet inflation/deflation
would minimise the chance of wound complications associated with oxidative
stress (Soneja et al., 2005; Estebe et al., 2011).
Figure 2.4: Illustration of potential reactive oxygen species (ROS) production
during ischemia-reperfusion. Abbreviations: O2•-
- superoxide anion; NO – nitric
oxide; ONOO- - peroxynitrite. Adapted from Powers et al. (2010).
2.2.3 Glutathione and Ischemia-Reperfusion
As mentioned in section 2.1.3.1, glutathione is a key endogenous thiol protein
intended to neutralise free radical molecules (Halliwell and Gutteridge, 2007).
The free radical production induced via ischemia-reperfusion has been measured
frequently in IRI studies to assess the redox disruption. Tissue homogenates
Endothelial
Cells
Muscle
Fibre
Capillary
Lumen
O2•-
NO
ONOO-
Nitric Oxide Synthase Mitochondria
O2•-
Xanthine Oxidase
Leukocyte
O2•-
27
(muscle, liver) are more commonly measured in rodent models. Puntel et al.
(2013) noted a reduction in GSH/GSSG ratio following 3 hrs of ischemia and 2
hrs of reperfusion in rat gastrocnemius muscle. A reduction in GSH
concentrations alone have also been reported post 4 hrs ischemia and 2 hrs
reperfusion in rat gastrocnemius muscle (Avci et al., 2012). During aorta-biformal
bypass surgery on elective patients, GSH concentrations were noted to
significantly decrease 24 hrs after initial reperfusion from ischemia (median ±
range; 113 ± 21) in comparison to preoperative levels (Westman et al., 2006).
Surprisingly, the ratio of GSH/TGH was not significantly different from
preoperative levels, as augmented oxidative stress would be expected to create
greater concentrations of GSSG, diminishing the GSH/TGH ratio (Halliwell and
Gutteridge, 2007). Although this may be due to extracellular exportation of GSSG
(Ballatori et al., 2009).
The literature collectively presents the disruption caused to the localised tissue
concentrations of GSH via ischemia-reperfusion. However, controversy exists in
relations to glutathione disruption in blood sampling. A significant increase in
GSSG was noted in systemic blood taken from the antecubital region at 3 and 10
min in reperfusion after TKR surgery (Garcia-de-la-Asuncion et al., 2012).
Interestingly, whole blood samples obtained from the operated knee also
displayed an increase in GSSG but at much greater concentrations. This disparity
between observed GSSG/GSH at localised and systemic sites has also been noted
by Karg et al. (1997). The authors demonstrated a significant increase in the
whole blood GSSG/GSH ratio at 5 min reperfusion in the operated leg when
compared to samples obtained from the arm. Finally, whole blood sampled at the
28
site of ischemia in knee surgery patients (femoral vein) acquiesced a significant
change in whole blood GSH and GSSG in comparison to sampling from a
systemic source (antecubital vein) (Mathru et al., 1996). Research from Garcia-de-
la-Asuncion et al. (2012), Karg et al. (1997) and Mathru et al. (1996) indicate that
the site of blood sampling can affect the yielded concentrations of glutathione
from a bout of ischemia-reperfusion. Therefore, GSH/GSSG concentrations will
provide a useful insight into redox changes induced via the hypoxic intervention
following TKR specific tourniquet application.
2.2.4 Ischemia-Reperfusion and Macromolecule Damage
The large oxidative burst initiated by reperfusion has a detrimental effect upon
localised tissue damage once the antioxidant defence is overwhelmed. Increased
levels of lipid peroxidation (4-HNE) were observed after 3 hrs of ischemia and
only 5 min of reperfusion in murine hind limb (Adachi et al., 2006). Hori et al.
(2013) found increased concentrations of MDA at 24 hrs reperfusion following
1.5 hrs of ischemia in both rat gastrocnemius and anterior tibialis. Furthermore,
increased levels of TBARS were observed at 10 min reperfusion in a clinical
population undergoing lower-leg extremity surgery (mean ± SD; 78.7 ± 13.3 min
of tourniquet inflation) when compared with baseline figures (Van et al., 2008).
However, as previously mentioned, TBARS are not specifically generated via LP
(Halliwell and Whiteman, 2004), therefore the results should be interpreted with
caution. Interestingly, ischemia alone induces lipid peroxidation, and is
exacerbated further upon reperfusion (Paradies et al., 1999). Greater lipid
peroxidation was also noted in hepatic (Lee et al., 2000) and brain tissue
(Sakamoto et al., 1991) post ischemia-reperfusion.
29
The deteriorated integrity of the cellular membrane from the reperfusion
associated oxidative attack leads to intracellular protein degradation (Halliwell
and Chirico, 1993). Similar to lipid peroxidation, the literature displays decisive
evidence for the role of ischemia-reperfusion in increasing protein oxidation. PC
concentrations were elevated in rat gastrocnemius muscle following 4 hrs of
ischemia and 2 hrs reperfusion in comparison to a SHAM condition (Avci et al.,
2012). These results were collaborated by Ozyurt et al. (2006) who noted a rise of
rat gastrocnemius PC concentration in the ischemia-reperfusion group when
analysed against a control. Conversely, Ozkan et al. (2012) found no significant
difference in PC concentrations in rat tibialis anterior between control and
ischemia-reperfusion condition subsequent to 3 hrs ischemia and 15 min
reperfusion. However, the ischemic-reperfusion condition did display a trend for
increased PC concentrations in comparison to control and may be due to a fairly
low participant number (n = 6 control, n = 10 ischemia-reperfusion).
The evidence outlined above demonstrates ischemia-reperfusion as a key player in
macromolecule damage and that varied durations of ischemia (1.5 – 3 hrs) all
provoke disruption in cellular structures. Thus, assessment of macromolecule
damage would provide a useful marker in measuring consequent effects of
improvements in redox disruptions following the hypoxic intervention.
2.2.4.1 Apoptotic Cell Death
Excessive macromolecule damage via augmented free radical production
commences activation of the intrinsic apoptosis pathway, through the initiation of
energy-dependant cascades (Elmore, 2007). Oxidative stress mediated DNA
30
damage stimulates the tumor suppressor protein, p53, to initiate either DNA repair
or, stimulate the apoptotic cascade via trans-activation of the large Bcl-2 family
(Elmore, 2007; Kroemer et al., 2007). The Bcl-2 protein group consist of both
pro- (Bax, Bak, Bid) and anti-apoptotic (Bcl-2, Bcl-XL, Bcl-x) members and are
maintained in a hierarchical model (Liu et al., 2010). P53’s induction of Bax
motivates the pro-apoptotic protein to bind to the mitochondrial outer membrane
inducing mPTP, either alone or with other Bcl-2 pro-apoptotic members, thus
allowing translocation of very large molecules (Kuwana et al., 2002). The large
pore alters the permeability of the mitochondrial membrane, disrupting ATP
synthesis and inducing an influx of Ca2+
, releasing cytochrome c subsequently
promoting the formation of the caspase-activating Apaf-1 apoptosome (Crompton,
1999; Hill et al., 2004). Interestingly, Ca2+
influx may not be directly accountable
for mitochondrial dysfunction as inhibition of calpain (downstream Bid cleaver)
offers myocardial protection from IRI (Chen et al., 2002). The apoptosome
triggers caspase-9 to become active, initiating caspase 3 leading to eventual
cellular organelle proteolysis, DNA fragmentation and decisive phagocytosis of
the apoptotic bodies (Elmore, 2007).
There is an array of evidence to link ischemia-reperfusion with cellular apoptosis.
Ex-vivo rat hearts participated in 15 min ischemia and 60 min reperfusion,
demonstrating large levels of apoptotic cell death in comparison to control and
that blockade of oxidative stress upon reperfusion diminished programmed cell
death (Maulik et al., 1998). These results have also been collaborated by Galang
et al. (2000). Galang and colleagues (2000) showed a dramatic increase in
apoptotic ex-vivo myocardiocytes after 30 min ischemia and 120 min reperfusion
31
in comparison to control and that the addition of SOD diminished this negative
consequence. However, apoptosis is an energy-dependant process and prolonged
exposures to ischemia can lead to vast ATP depletion (Kim et al., 2003; Elmore,
2007). If enough of the cells’ mitochondria are damaged, necrotic death will ensue
rather than the favourable apoptosis (Yang et al., 2010). Nevertheless, there is no
precise feature for either apoptosis or necrosis as both share similar mechanistic
pathways, invariably creating difficulty when distinguishing between the two
(Jaeschke and Lemasters, 2003).
Apoptosis is cited to play a role in the pathophysiology of skeletal muscle cell loss
(Dirks and Leeuwenburgh, 2002) and occurs during ischemia reperfusion
following cellular lipid peroxidation (Maulik et al., 1998). Therefore, an
intervention which could attenuate apoptosis during tourniquet mediated IRI may
enhance recovery times post TKR surgery.
2.2.4.2 Necrotic Cell Death
Necrosis is characterised by cytoplasmic bulging, plasma membrane rupture and
organelle damage, resulting in inflammation due to release of cellular components
(Festjens et al., 2006). Tumor necrosis factor-α (TNF-α) produced primarily from
activated macrophages can induce either cell survival or cell death depending on
the cellular environment via ligand-binding with death receptors, TNF-R1 and
TNF-R2 (Chen and Goeddel, 2002; Festjens et al., 2006). Although necrosis and
apoptosis have very different outcomes, both can be initiated by TNF-α,
depending on caspase inhibition (Vanden Berghe et al., 2004). TNF-α cascade
signalling induction of Fas-associated death domain mediates apoptosis (Chen et
32
al., 2002), while receptor-interacting protein triggers necrotic cell death (Hsu et
al., 1996).
TNF has been demonstrated to provoke skeletal muscle injury during ischemia-
reperfusion, since antibody blockade of TNF resulted in a reduction in skeletal
muscle injury (Gaines et al., 1999). This result is in collaboration with Seekamp et
al. (1993) who noted a reduction in skeletal and lung injury through TNF
obstruction. However, TNF blockade by Sternbergh et al. (1994) did not present a
reduction in skeletal muscle endothelial injury, suggesting that TNF may not play
a primary role in IRI injury. Interestingly, post TKR surgery, only moderate rises
in cytokine concentration (TNF, interlukin-1) have been observed after 2 hrs
reperfusion (Clementsen and Reikeras, 2008), although this moderate
accumulation of cytokines is in disagreement with Seekamp et al. (1993). The low
levels of cytokine observed by Clementsen and Reikeras (2008) in comparison to
Seekamp and colleagues (1993) may in part be due to the large disparity between
ischemic durations (78-125 min and 4 hrs respectively).
Skeletal muscle necrosis via IRI has been cited to affect muscle function through
decreased muscle twitch and contractual force (Kearns et al., 2001), thus an
attenuation of this muscle detriment could improve the surgical outcome of TKR
patients.
2.2.4.3 Toll-Like Receptors
Necrotic signalling transduction is not just limited to the death receptor pathway,
toll like receptors (TLRs) have also been implicated in invoking IRI. TLRs are a
family of leucine rich transmembranal proteins involved in regulating innate and
33
adaptive immune response to pathogenic invaders (Takeda et al., 2003). TLRs are
present on immune cells, such as macrophages (Hadley et al., 2007) and non-
immune cells including skeletal muscle (Frost et al., 2006). TLRs have been
implicated to participate in the pathophysiology of IRI (Arumugam et al., 2009;
Khandoga et al., 2009) with TLR-2 (Favre et al., 2007) and TLR-4 (Kaczorowski
et al., 2007) implicated as major participators. HSPs and high mobility group box-
1 proteins among others, are considered ligands for TLR-2 and TLR-4, which are
secreted into the surrounding environment from stimulated leukocytes (Park et al.,
2004; Arumugam et al., 2009). The stimulation of TLRs induce numerous
intracellular signalling cascades via mitogen activated protein kinase (MAPKs)
and IκB kinase, which are very similar to the interlukin-1 pathway resulting in the
stimulation of pro-inflammatory transcription factors such as nuclear factor-κB
(NF-κB) and activator protein 1 (AP-1) (Takeda et al., 2003; Arumugam et al.,
2009). The consequential activation of NF-κB and AP1 induces a plethora of
functions including a greater expression of cytokines, adhesion molecules and
chemokines (Batra et al., 2011).
Research into TLR-2(-/-)
knockout mice has been shown to abolish endothelial
dysfunction when compared with wild type mice in cardiac tissue after 30 min
ischemia and 1 hr reperfusion (Favre et al., 2007). The reduction in endothelial
dysfunction was attributed to a reduction in ROS production and leukocyte
infiltration. In fact, this explanation was confirmed by Khandoga et al. (2009).
The authors utilised TLR-2(-/-)
knockout mice with the addition of mice with
mutant TLR-4 receptors and demonstrated that after ischemia-reperfusion a
reduction in neutrophil endothelial migration was noticed in comparison to mutant
34
TLR-4 receptor mice, in addition both TLR-2(-/-)
knockout and TLR-4 mutant
mice displayed attenuated vascular leakage. Indeed, the use of circulatory TLR-2
antibody inhibitors reduced infarct size and diminished functional capacity in
murine hearts post ischemia-reperfusion (Arslan et al., 2010). This suggests that
TLR-2 plays an important role with trans-endothelial migration of leukocytes in
IRI.
Similar results in IRI were also observed in TLR-4(-/-)
knockout mice (Oyama et
al., 2004) and blockade of TLR-4 via eritoran (Shimamoto et al., 2006). Although
both TLR-4 and TLR-2 appear to be in part responsible for the innate
inflammatory response during IRI, the latter is illustrated to have a greater role in
neutrophil migration (Khandoga et al., 2009).
The culmination of the factors above, in addition to the tissue hypoxia, could
negatively affect wound healing following prolonged tourniquet use (Estebe et al.,
2011). Therefore, interventions are required to attenuate the duration of wound
healing associated with tourniquet use in TKR surgery.
2.3 Ischemic Pre-Conditioning
To avoid the deleterious effects of IRI multiple strategies have been proposed.
Numerous studies have administered supplements to mitigate the damage
sustained via IRI (Nedrebo et al., 2003; Ozyurt et al., 2006; Avci et al., 2012; Hori
et al., 2013). However, pharmacological interventions must be timed perfectly, as
well as administered in correct doses with regards to intravenous administration,
to ensure the drug has reached the tissue prior to the ischemic insult and in a
significant quantity (Wang et al., 2002). In addition, rigorous testing of novel
35
pharmaceuticals is required to avoid fatal interactions with anaesthesia (Weldon-
Bellville, 1972). Furthermore, many of the trials used for supplementation
administration are performed on healthy individuals and the effect of
pharmaceuticals upon patients who are morbidly obese (often associated with
diabetes type II, cardiovascular disease, sleep apnoea) is not well understood
(Wang et al., 2002; Samson et al., 2010). Interestingly, morbidly obese is
considered an independent cause of knee osteoarthritis (Samson et al., 2010) and
morbidly obese individuals contribute to around 28% of total primary TKR
patients (Dehn, 2007), thus a non-pharmaceutical intervention would be
beneficial.
In contrast to supplementation, a landmark study conducted by Murry et al. (1986)
involved a group of dogs receiving 4 cycles of 5 min coronary occlusion and 5
min reperfusion, prior to 40 min of solid coronary ischemia and a 4 day
reperfusion period. Surprisingly, the dogs who received the ischemic/reperfusion
cycles displayed a reduction in infract size in comparison to a control group only
receiving 40 min occlusion. This technique has been aptly termed ischemic
preconditioning (IPC) and since this discovery by Murry and colleagues (1986)
many researchers have pursued this phenomenon in various tissues other than
cardiac muscle, including hepatic (Yoshizumi et al., 1998), skeletal muscle (Saita
et al., 2002), brain (Dawson and Dawson, 2000) and renal tissues (Toosy et al.,
1999). Thus, to establish the extent of which the non-invasive hypoxic
preconditioning can attenuate tourniquet mediated oxidative stress, a comparison
to a previously used non-invasive technique is required.
36
The mechanism by which IPC protects tissue against IRI is not fully understood
(Yang et al., 2010), however, it has been established that the protective effects
appear in a biphasic pattern (Das and Das, 2008). The early phase is proposed to
last between 2-3 hrs (Yang et al., 2010), while a delayed phase appears 24 hrs
after the IPC protocol and can last up to 3 days (Hausenloy and Yellon, 2010)
(Figure 2.5).
Figure 2.5: Schematic depicting a list of potential mediators and the proposed
time frame for the protection offered by ischemic preconditioning. Abbreviations:
PKC – protein kinase C; ROS – reactive oxygen species; HSP70 – heat shock
protein 70; HSP32 – heat shock protein 32; COX-2 - Cyclo-oxygenase 2; RISKs –
reperfusion injury salvage kinases.
2.3.1 Early Phase
Early phase IPC protection has been linked to the activation of G-coupled
adenosine receptors via the release of endogenous adenosine during the brief
ischemia (Liu et al., 1991). Stimulated adenosine receptors main target during
Time Succeeding Ischemic Preconditioning (hrs)
0 3 24 72
Early phase of protection
No protection
Delayed phase of protection
Adenosine PKC
Bradykinin ROS
Antioxidant enzymes
HSPs COX-2
RISKs
Mediators of Ischemic Preconditioning
37
early IPC is the family of 12 serine/threonine kinases known as protein kinase C
(PKC) (Cohen et al., 2000). It has been hypothesised that stimulation of PKC
activates 5’-nucleotidase, generating larger concentrations of adenosine via
degradation of AMP, which is of abundance during ischemia (Kitakaze et al.,
1995). Additionally, PKC is thought to be initiated directly via ROS signalling as
intravenous administration of free radical scavengers removes the protection
granted from IPC (Baines et al., 1997), while cellular models have demonstrated
that the addition of oxidants promotes preconditioning (Vanden Hoek et al.,
1998).
Mitchell et al. (1995) demonstrated that PKC activator, 1,2-diacylglycerol (DAG)
produced similar cardio-protective effects to IPC in ex-vivo rat hearts. Moreover,
blockade of PKC abolished the protective effect offered from IPC. The negated
effects of IPC were also noted by Ytrehus et al. (1994) following PKC blockade
in rabbit hearts. However, the specific PKC isoenzyme that contributes to IPC is
still in debate. PKC-δ(-/-)
knockout mice displayed augmented ischemic tissue
damage following IPC (Mayr et al., 2004). Conversely, Bright et al. (2004) noted
that inhibition of PKC-δ reduced cerebral IRI in rats, indicating the equivocal
nature of PKC-δ in inducing IPC. However, it appears that PKC-ε is essential for
IPC protection (Ping et al., 2002), and that knockout mice deficient in PKC-ε
displayed inhibited protection afforded from IPC (Saurin et al., 2002).
Consequently, PKC-δ may not be essential in IPC unlike the isoform PKC-ε.
IPC is proposed to act on the mPTP, offering protection through maintaining
inhibition of pore opening (Das and Das, 2008). As previously mentioned, (in
38
section 2.2.4.1) opening of the mPTP has catastrophic effects upon the
mitochondria, with eventual apoptosis ensuing. Mitochondrial potassium ATP
(mKATP) channel activation has been linked to inhibiting mPTP opening, although
the mechanisms involved are unclear (Hausenloy et al., 2002). The activation of
PKC-ε via adenosine allows the stimulated serine/threonine kinase to open the
mKATP channel (Hu et al., 1999). Influx of K+ into the mitochondria maintains
Ca2+
concentrations via reducing the electrochemical gradient for Ca2+
movement
into the mitochondria (Holmuhamedov et al., 1999) thus restraining Ca2+
mediated mPTP opening and subsequent apoptosis. It has also been speculated
that PKC-ε may regulate the apoptosis through coimmunoprecipitation to
components of the mPTP (voltage dependant anion-selective channel, adenine
nucleotide translocase) inhibiting mPTP opening (Yonekawa and Akita, 2008).
Therefore, any immediate reduction in oxidative stress markers from IPC to a
subsequent bout TKR specific tourniquet application would involve the
mechanisms outlined above.
2.3.2 Delayed Phase
After the initial 2 hr window of protection offered by IPC, the delayed phase takes
effect 24 hrs post preconditioning and has been attributed to altered gene
expression and de novo synthesis of proteins (Carden and Granger, 2000).
Mediators of delayed protection are generally protein kinases which are activated
during the IPC stimulus resulting in activation of transcription factors (Hausenloy
and Yellon, 2010).
39
The established mediator of early protection, PKC, has also been implicated to
produce delayed protection (Qiu et al., 1998). Qiu et al. (1998) demonstrated
inhibition of PKC after the ischemic stimulus did not abolish the protection
offered by IPC suggesting crucial activation of PKC occurs during the ischemic
bout. Qiu and colleagues (1998) also established that general activation of PKC
without ischemia mimicked the effects of delayed IPC. MAPKs have also been
recognised as delayed protection mediators via upstream stimulation from PKC
(Xuan et al., 2005). Indeed, research by Fryer et al. (2001) noted the integral role
of extracellular signal regulated kinase (ERK) and p38 MAPK in delayed
cardioprotection. The stimulation of mediators initiates activation of a variety of
cell stress signalling transcription factors associated with delayed IPC such as
hypoxia induced factor -1α (Xi et al., 2004), NF-κB (Xuan et al., 1999) and AP-1
(Li et al., 2000). Although, it has been suggested that transcription factors can be
recruited differentially and still induce the requisite delayed protection
(Hausenloy and Yellon, 2010). Cell stress transcription factors can be induced via
a differing stress (e.g. hyperthermia) but still yield protection from IRI,
demonstrating a cross-tolerance effect (Horowitz et al., 2004). Therefore, hypoxic
preconditioning could yield delayed protection from ischemia via stressor cross-
tolerance.
2.3.2.1 Antioxidant enzymes
Transcriptional mediators of delayed IPC are proposed to up-regulate antioxidant
enzymes (Hausenloy and Yellon, 2010), including, peroxisomal catalase,
glutathione peroxidase and MnSOD (Das et al., 1993). MnSOD is a key
mitochondrial protein which is synthesised in the cytoplasm and imported
40
posttranslationally into the mitochondria, where it functions to catalyse the
dismutation of O2•-
into H2O2 and molecular oxygen (Jin et al., 2005). Dramatic
increases in MnSOD have been noted 24 hrs following a bout of IPC in canine
hearts (Hoshida et al., 1993). Indeed, Zhou et al. (1996) also demonstrated greater
MnSOD induction and activity 24 hrs following IPC in rat myocytes in
comparison to control. However, not all research has verified increases in
MnSOD activity succeeding IPC. Tang et al. (1997) showed that delayed IPC did
not increase MnSOD activity in porcine hearts. Nevertheless, the discrepancy in
results observed by the various research groups may be attributable to specie or
IPC protocol differences. Regardless of these variances, it is likely that an
increase in MnSOD following IPC would partially augment protection from future
ischemic insults.
2.3.2.2 Heat shock protein 72
Marber and colleagues (1993) were the first to illustrate an increase in HSP72
following IPC (4 x 5 min ischemia interrupted with 10 min reperfusion) 24 hrs
earlier in cardiac tissue, with the rise in HSP72 associated with the mechanisms
involved in delayed preconditioning. HSPs are a family of highly conserved
cytoprotective proteins ubiquitously found in cells, which function as molecular
chaperones facilitating folding and transportation of newly synthesised and
denatured proteins (Morton et al., 2009). In addition, HSPs play a versatile role in
cellular survival via interaction with molecules associated with the apoptotic
cascade (Kalmar and Greensmith, 2009). HSPs are defined by their estimated
molecule weight and tend to be grouped into sub-group families (i.e. HSP70 (70
kDa)), and are localised throughout various regions of the cell (Morton et al.,
41
2009). HSP70 in particular, is highly inducible during stressful situations,
rescuing damaged proteins and maintaining cellular protein synthetic capacity
(Noble et al., 2008).
The coordinated organisation of activated HSP gene expression (Hsp) in response
to a variety of stressors, including ischemia (Chang et al., 2001), hyperthermia
(Oishi et al., 2003) and hypoxia (Taylor et al., 2010) is called the heat shock
response. Under normal physiological conditions, HSP70 is bound to its
transcription factor, heat shock factor 1 (HSF1), in an inactive monomeric state
(Abravaya et al., 1992). However, an external stressor induces intracellular
protein unfolding and denaturation, stimulating the dissociation of the HSP70 and
HSF1, through HSP70 preferential binding to the damaged protein (Noble et al.,
2008). Unbound, HSF1 undergoes trimerisation with other free HSF1,
subsequently binding to the heat shock element in the promoters of heat shock
genes, allowing transcription of additional HSPs (Sarge et al., 1993) (Figure 2.6).
The integral role of HSP72 during cellular stress and its rapid induction via a
variety of stressful stimuli make it an excellent marker of redox disturbance
(Taylor et al., 2012). Indeed, oxidative stress has been cited to up-regulate HSP72
via signal activation of HSF1 and by directly oxidising cellular proteins (Morton
et al., 2009). Therefore, a rise in Hsp72 would indicate greater cellular stress
following the TKR specific tourniquet application.
42
Figure 2.6: Stress induced activation of the heat shock response mechanism.
Unfolded proteins induce the dissociation of HSP70 from its bound monomeric
state with HSF1, allowing HSP70 to aid in the refolding of damage proteins.
Unbound HSF1 trimerises with other free HSF1, subsequently binding the heat
shock element, initiating transcription. Abbreviations: HSP70 – heat shock protein
70; HSF1 – heat shock factor 1. Adapted from Nobel, Milne & Melling (2008).
Since the original observation between delayed IPC and HSPs by Marber et al.
(1993) subsequent research groups have explored this connection. Okubo et al.
(2001) injected recombinant adenovirus encoding for HSP70 into in-vivo rabbit
hearts 4 days prior to 30 mins cardiac ischemia and 3 hrs reperfusion. The authors
Functional Protein
HSP70
HS
P70
HSP mRNA
HS
F
1
HS
F
1 H
SF
1
Unfolded Protein
Protein Re-folding
HSF1 HSP70 HSP70
HS
F1
HS
F1
HS
F1
HS
F1
Heat Shock Element
Functional Protein
43
noted a reduction in infarct size in the HSP70 vector comparison to a saline
injection alone. This evidence for the role of HSP70 in delayed IPC was also
observed by hyperthermic preconditioning (Lepore et al., 2000). Lepore and
colleagues (2000) performed hind limb heating at 42°C for 20 min prior to 2 hrs
ischemia and 24 hrs reperfusion. The passive heating bestowed a rise in HSP72
and subsequent protection from the ischemic insult. Interestingly, Tanaka et al.
(1998) observed increases in HSP70 after just 3 hrs following IPC in rabbit hearts,
with concentrations remaining detectable by immunohistochemical techniques for
up to 72 hrs. In contrast to this, Lepore and Morrison (2000) did not observe an
increase in HSP70 gene expression from 2 x 10 min bouts of ischemia with 15
min reperfusion interruptions in rat hind limb skeletal muscle. Additionally, the
authors noted that there was not a significant increase in viable fibres following 2
hrs of ischemia in comparison to a control group. However, the poor sample
number (n = 3) and altered preconditioning protocol to previous successful work
(Marber et al., 1993) may have led to the non-significant result (p = 0.16).
Interestingly, repeated bouts of endurance exercise can provide a marked rise in
HSP72 within cardiac tissue (Powers et al., 1998; Demirel et al., 2003) inducing
protection from IRI (Steel et al., 2004). However, HSP72 is not necessarily
required to attenuate IRI. Research from Taylor et al. (1999) and Hamilton et al.
(2001) both demonstrated that exercise bestowed protection from IRI without an
increase in cardiac HSP72. It is possible that the disparity between cardiac and
skeletal muscle research may be attributable to varying degrees of HSP72
dependency in differing tissues. Indeed, Hamilton et al. (2001) postulated that
protection in cardiac tissue mediated via exercise may be partly due to greater
44
antioxidant capacity. It could therefore be speculated that cardiac tissue relies
more heavily on antioxidant enzymes in comparison to skeletal muscle which may
depend more on HSPs, although further research is necessary.
In summary, HSP72 appears to play a key role in skeletal muscle delayed IPC
(Marber et al., 1993; Okubo et al., 2001); therefore a deferred increase in Hsp72
from IPC may indicate the onset of delayed preconditioning.
2.3.2.3 Heat shock protein 32
HSP32 (or Heme oxygenase-1) is another member of the cytoprotective family
associated with delayed IPC (Hausenloy and Yellon, 2010). Free heme is bound in
hemoproteins during homeostasis, however, during oxidative stress, free heme
molecules are released from heme pockets within the hemeproteins and in doing
so produce ROS via Fenton chemistry (Gozzelino et al., 2010). HSP32 is an
evolutionary conserved enzyme that catabolises the ROS producing free heme
into biliverdin, carbon monoxide (CO) and iron, (Tenhunen et al., 1968).
Bilverdin is then available for enzymatic conversation via bilverdin reductase
(BVR) into the cytoprotective anti-oxidant bilirubin (Clark et al., 2000).
HSP32 expression is regulated by numerous stress-associated effectors such as
heme, hyperthermia, and hypoxia (Alam and Cook, 2007). Transcriptional
activation of HSP32 is controlled by a variety of transcription factors including,
nuclear factor E2-related factor 2 (NrF2), NF-κB, AP1 and HSF-1 (Kim et al.,
2011). Stressful circumstances have the ability to stimulate multiple transcription
factor groups simultaneously, with differing group’s regulating slightly different
aspects in HSP32 stress response (Alam and Cook, 2007). The MAPK cascade
45
appears to be the foremost mediator of HSP32 gene regulation (Figure 2.7),
involving the three major sub-families, ERK, c-Jun N-terminal kinases (JNKs)
and p38 kinases (Alam et al., 2004). Indeed, ERK, JNKs and p38 MAPK were all
attributed to the upregulation of HSP32 following IRI (Zhang et al., 2002).
Figure 2.7: Regulation of the HSP32 gene via the major stress-response
transcription factors. Stimulation of transcription factors occurs through
MAPK signalling (direct phosphorylation (solid line); signalling cascade
(dotted line)) or directly by stressful stimuli (grey line). Abbreviations: NrF2 –
Nuclear factor E2-related factor 2; NF-κB – Nuclear factor-κB; AP1 –
Activator protein 1; HSF1 – Heat Shock factor 1; MAPK – Mitogen activated
protein kinases; HSP32 – Heat shock protein 32. Adapted from Alam & Cook
(2007).
HSP32 Gene
NrF
2
AP
1
HSF
1
NFκB
MAPK Signalling
MAPK Signalling
Redox Disturbance
Inflammatory
Mediators
Protein
Denaturation Cell growth/death
signals
HSP32 mRNA
46
Similar to HSP72, the inducible form of HSP32 has also been demonstrated to be
a marker of oxidant mediated cell damage (Rothfuss et al., 2001). Thus, an
increase in Hsp32 following tourniquet application, would indicate a rise in
oxidative stress.
Myocardial cells undergoing IPC displayed an increase in HSP32 24 hours post
ischemic stimulus, in addition it to an increase in cell viability in comparison to a
simulated ischemic group (Jancso et al., 2007). Further evidence for HSP32’s
induction through IPC was noted by Zeynalov et al. (2009), who performed IPC
on wild type mice and found a significant increase in protein expression 24 hours
post stimulus in comparison to a sham control. Furthermore, the anti-oxidant by-
product of HSP32 activity, bilirubin, was noted to be significantly increased in
IPC rat skeletal muscle in contrast to control (Badhwar et al., 2004).
2.4 Hypoxic Preconditioning
HPC has been utilised previously to bestow cellular tolerance as a stratagem to
avoid deleterious effects from disturbances to the redox balance (Taylor et al.,
2012). Moreover, hypoxic exposure has been described to have similar protective
effects as IPC in providing tolerance to a subsequent sustained bout of ischemia
(Beguin et al., 2005). In line with IPC, HPC also offers biphasic protection at
similar intervals (i.e. within 2 hrs and between 24-72 hrs of stimulus removal)
(Zhao et al., 2013). Furthermore, both IPC and HPC appear to share similar redox
pathways in response to hypoxia mediated oxidative stress (Zuo et al., 2013).
PKC-ε is attributed to an integral role in inducing early IPC, but the kinase has
additionally been demonstrated to be up-regulated following HPC and that general
47
blockade of all PKC isoforms abolish the protection conferred by the hypoxic
exposure (Berger et al., 2010). In addition, it has been shown that the increase in
ROS via HPC initiates ischemic tolerance (Vanden Hoek et al., 1998), perhaps via
ROS mediated stimulation of PKC. Indeed, PKC-ε has also been shown to up-
regulate HSP32 (Ryter et al., 2006), further elucidating the vital role of PKC-ε in
HPC. Thus, the HPC undertaken in the present thesis may offer protection to
tourniquet mediated oxidative stress immediately following the hypoxic exposure.
The delayed phase of HPC operates in an identical manner to IPC by initiating de
novo synthesis of proteins (Zhao et al., 2013). HPC has been speculated to
activate the transcription factor, hypoxia induced factor -1α, which stimulates up-
regulation of target genes including, vascular epidermal growth factor, inducible
nitric oxide synthase and erythropioetin (Ran et al., 2005). These genes would
promote greater blood flow and oxygen delivery for subsequent ischemic insults
(Ran et al., 2005). Moreover, Taylor et al. (2010) noted an increase in monocyte
HSP72 gene expression following a 75 min period of normobaric hypoxia (14.5%
O2) in vivo. Indeed, HPC mediated up-regulation of HSP72 has been
demonstrated to diminish the deleterious effects of IRI in the rat kidney (Yeh et
al., 2010). Furthermore, HPC has been elicited to increase HSP32 following a
prolonged hypoxic exposure, attenuating hepatic IRI in rats (Lai et al., 2004).
The mechanisms outlined above describe the process in which HPC could
potentially protect skeletal muscle against IRI following TKR specific tourniquet
application. Therefore, a reduction in redox mediated tissue damage could lead to
less wound complications and greater surgical outcomes (Soneja et al., 2005;
Estebe et al., 2011).
48
Mice kept in normobaric hypoxic (10% O2) chambers for 4 hrs followed by 24 hrs
in normoxia prior to 20 min global cardiac ischemia and 30 min reperfusion,
displayed a reduction in infarct size in comparison to control mice in isolated
hearts (Xi et al., 2002). Furthermore, HPC prior to 6 hrs of hypoxia (0.5% O2) and
12 hrs re-oxygenation provided a significant reduction in apoptosis of
mesenchymal cells in-vitro (Wang et al., 2008). Further in-vitro work by Wu et al.
(2013) illustrated that 20 min of HPC simulated by incubation in anoxia (95 % N,
5% CO2) 24 hrs prior to 3 hrs of anoxia induced a reduction in ECs apoptosis.
Animal in-vivo research by Berger et al. (2010) performed 4 hrs of systemic
normobaric hypoxia at different concentrations (8%, 12% and 16% O2) in rats.
The author noted a significant reduction in infarct size in all HPC condition in
comparison to control.
In summary, IPC has been shown on numerous occasions to diminish the
deleterious effects of IRI in a multitude of tissues, with similar results observed in
HPC. However, there is a lack of research regarding the effects of HPC in human
in-vivo skeletal muscle and circulation following TKR specific tourniquet
mediated limb ischemia.
49
Chapter 3: Methodology
50
3.1 Participants
Eighteen recreational healthy male participants were recruited for the study (see
Table 3.1 for demographic data). Prior to experimental contribution, participants
were all informed of the procedure and the risks involved in participating,
subsequently providing written informed consent, according to the principles set
out in the Declaration of Helsinki (Appendices B - E). Each participant was
medically screened and ethical approval was obtained via the University of
Bedfordshire’s, Sport and Exercise Science Department Human Ethics
Committee. It was mandatory that participants were free from any
musculoskeletal injury or acute or chronic sickness, in addition to abstinence from
medication (vitamin supplementation), ergogenic aids (creatine, ß-alanine,) and
extreme environments (hyperthermia, hypoxia) that may affect any variables
intended to be measured in this study. All participants were non-smokers and 72
hours prior to testing, participants were required to refrain from alcohol, caffeine
and exercise (the full list is available in Appendix A). Participants were randomly
allocated into either control (SHAM), hypoxic preconditioning (HYP) or ischemic
preconditioning (TOR) conditions.
3.2 Anthropometric Data
Body mass (kg), and height (m) were assessed with the use of a Digital Tanita
scales (BWB0800, Allied Weighing, UK) and a wall-mounted Stadiometer
(Holtain Ltd, UK) respectively. Participants were asked to refrain from fluid/food
ingestion 4 hours, in addition to an evacuated bladder immediately prior to
estimation of percentage muscle and fat mass (%) through utilisation of air
51
pletysmography (BodPod 2000A, Cranlea, UK). Furthermore, individual’s blood
pressure was noted in triplicate and an average was obtained (M5-I, Omron,
Cranlea, UK). Participants were seated for 5 min and requested to close their eyes
and relax prior to measurement acquisition based on the manufacturer’s
instructions. Finally, thigh circumference was obtained in triplicate using a tape
measure (Body Care, HaB Direct, UK) at 40% of the distance from the knee joint
centre to the greater trochanter on the right leg (Table 3.1).
52
Table 3.1: Participant demographic data
Measure SHAM HYP TOR
Mean SD Range Mean SD Range Mean SD Range
Age (years) 22.2 2.9 18 - 26 20.8 2.4 19 - 25 18.5* 0.6 18 - 19
Height (m) 1.83 0.06 1.75 - 1.92 1.77 0.10 1.67 - 1.93 1.79 0.04 1.73 - 1.83
Mass (kg) 80.4 12.4 62.8 - 93.4 73.5 8.7 61.9 - 86.6 76.7 7.8 64.8 - 87.4
Lean mass (%) 85.8 3.5 79.3 - 88.5 84.9 5.3 78.9 - 91.4 86.5 3.0 81.5 - 90.1
Fat mass (%) 14.2 3.5 11.5-20.7 15.3 5.5 8.6 - 22.1 13.5 3.0 9.9 - 18.5
Thigh Circumference (cm) 44.7 2.6 40 - 47 42.8 2.1 40 - 46 43.3 2.4 39 - 46
Systolic Blood Pressure
(mmHg) 124.0 3.0 120 - 129 125.8 1.9 123 - 129 125.5 2.1 123 - 129
Diastolic Blood Pressure
(mmHg) 76.2 7.0 65 - 83 75.0 6.0 65 - 81 79.7 8.0 70 - 92
* Significant difference v.s. SHAM (p < 0.05)
53
3.3 Hydration Status Assessment
Dehydration has the potential to induce oxidative stress (Paik et al., 2009), which
in turn initiates regulation of HSPs (Ahn and Thiele, 2003). Therefore, the author
felt it necessary to assess hydration status prior to commencing the trials in order
to minimise erroneous results. Urine refractormetry has been previously shown to
provide a reliable measure for urine specific gravity to assess hydration status
(Stuempfle and Drury, 2003).
Upon arrival to the laboratory, participants were requested to provide a urine
sample for analysis via a urine refractometer (Pocket Pal-Osmo, Atago Vitech
Scientific, HAB Direct, UK). Samples were assessed in triplicate and
euhydratrion was accepted at 200-600 mOsmols∙kgH2O-1
utilised previously by
(Hillman et al., 2011)
3.4 Blood Collection
Blood samples were obtained using Vasoplus Needles (22G x 1 ½”, Grenier Bio-
One, UK) from the antecubital region via standard venepuncture techniques
(Figure 3.1). Samples were drawn directly into three separate vacuette containers
(Vacuette®, Grenier Bio-One, UK) treated with K3EDTA (HSP gene expression
(Hsp)), sodium citrate (TGSH, GSSG) or lithium heparin (PC).
54
The author originally proposed to acquire blood samples from the ischemic lower
leg, in addition to blood from the antecubital region, as previous research (Karg et
al., 1997; Garcia-de-la-Asuncion et al., 2012) has shown differing metabolite
concentrations in systemic and localised blood following limb ischemia.
However, following pilot-work, it was established that obtaining blood from the
lower leg was not possible and only systemic blood was collected thereafter.
Figure 3.1: Image of the venepuncture technique used during the study.
55
3.4.1 K3EDTA Treated Blood
Blood treated with the anti-coagulant EDTA has been demonstrated to increase
the yield of Hsp concentrations in comparison to alternative method (Whitham
and Fortes, 2006). Leukocytes were isolated utilising an adaptation of a technique
previously validated (Sandstrom et al., 2009; Taylor et al., 2010; Hillman et al.,
2011).
Briefly, 1 mL of K3EDTA blood was added to 1:10 eythrocyte lysis solution
(Miltenyi Biotec, UK) and allowed to incubate at room temperature for 15 min,
prior to isolation via centrifugation at 400 G for 5 min at 4°C. Supernatant was
removed and the remaining pellet was washed with 2 mL of phosphate buffered
saline (PBS) solution (Fisher Scientific, UK) then centrifuged at 400 G for 5 min
at 4°C. Supernatant was discarded and a repeat wash was performed. The pellet
was suspended in 1 mL of PBS and separated equally into two 1.5 mL RNase free
eppendorfs then centrifuged at 17 000 G for 5 min at 4°C. The remaining
supernatant was aspirated prior to the pellet being completely re-suspended in 200
µL of TRIzol reagent (Sigma Aldrich, Dorset, UK) and stored at -80°C for
subsequent RNA extraction (section 3.10).
3.4.2 Sodium Citrate Treated Blood
Two mL of sodium citrate blood was immediately added to 8 mL of freshly
prepared 5% metaphosphoric (Sigma Aldrich, Dorset, UK) and left to incubate on
ice for 15 min before being centrifuged at 12 000 G for 15 min at 4°C. The
clarified supernatant was collected and separated into 1.5 mL eppendorfs prior to
56
storage at -80°C until future analysis for TGH and GSSG with commercially
available kits (section 3.9).
3.4.3 Lithium Heparin Treated Blood
The collected blood was immediately centrifuged at 900 G for 10 min at 4°C
before the plasma was separated into 1.5 mL eppendorfs and stored at -80°C until
future analysis via commercially available kits (section 3.8).
3.5 Muscle Biopsies
Muscle Biopsies were obtained from the lateral head of the gastrocnemius of the
ischemic leg 2 cm apart under local anaesthetic (2% lidocaine hydrochloride),
specifically avoiding the fascia of the muscle as outlined by Trappe et al. (2013),
using a disposable biopsy needle (Figure 4.2) (12 x 16, Disposable Monopty Core
Biopsy Instrument, Bard Biopsy Systems, USA) and placed into 2 mL RNase free
tubes (detailed method in Appendix F). Multiple sample sites were elected as
previous research has shown multiple passes from a single incision can alter tissue
gene expression (Friedmann-Bette et al., 2012). Samples collected (20-30 mg)
were immediately frozen in liquid nitrogen (-196°C) and stored at -80°C for
subsequent RNA extraction (see section 3.10). Serial muscle biopsies have been
previously demonstrated not to provoke stress proteins in the residual tissue
(Khassaf et al., 2001).
57
3.6 Experimental Design
Participants arrived at the University of Bedfordshire’s Sport and Exercise
Laboratories for two separate visits at 11:30. The laboratories were maintained at
a constant temperature (mean ± SD; 22 ± 1°C) throughout the entire experiment.
The first visit was used to provide anthropometric data as outlined in section 3.2
and occurred between 7-14 days prior to the second visit. Participants were
Figure 3.2: Images of the muscle biopsy procedure. (A) The
gastrocnemius was cleaned with cyclohexane, injected with lidocaine
subsequently a small incision was made. (B) 20-30 mg of tissue was
obtained using Bard Biopsy needle.
A
B
58
requested to consume a standardised meal in the evening prior to their second
visit.
Upon arrival to the laboratories for the second time, participants were requested to
provide a urine sample and abstinence information as depicted by section 3.3 and
3.1 respectively, in addition to providing a food diary for the past 3 days
ingestion. Participants were positioned in an inclined supine position throughout
the trial and asked to move as little as possible. Participants were permitted a
standardised breakfast and lunch 3 hrs prior to commencing the trial and 2 hrs into
experiment respectively. Compliance was monitored via a questionnaire. The
author would have preferred participants to be fasted for 6 hrs and be nil-by-
mouth for the duration of the trial to simulate hospital protocol. However, pilot-
work demonstrated that due to the invasive nature of the study, individual’s
suffered from bouts of syncope during the muscle biopsy procedure, thus making
this unethical.
All participants were initially rested for 1 hour prior to undertaking their allocated
40 min preconditioning intervention. Individuals in the SHAM condition received
an extended rest period. HYP inhaled 14.3% O2 (2, 980 m above sea level) in
normobaric pressure via an adjustable hypoxicator (Everest Summit II, The
Altitude Centre, UK), which has been shown to be an adequate stimulus to induce
a cellular stress response in-vivo (Taylor et al., 2010). The hypoxicator produces
the necessary hypoxic load via O2 filtration. During the hypoxic exposure,
participants heart rate (HR) and oxyhaemoglobin saturation were measured every
5 min via finger pulse oximetry (Onyx® II 9550, Nonin Medical, USA). TOR
59
received 4 cycles of 5 min ischemia and 5 min reperfusion at 100 mmHg above
the participant’s systolic pressure on their right leg. The chosen pressure was
based upon previous research by Estebe et al. (2000), with the duration of
ischemic cycles adapted from work by Koca et al. (2011). All ischemic bouts
throughout were produced via a straight 10 cm wide tourniquet cuff (AET, Anetic
Aid, Leeds, UK) positioned superiorly to cotton wool padding (Estebe et al.,
2011), with pressure maintained by means of an electronic tourniquet unit (AET,
Anetic Aid, Leeds, UK).
Upon cessation of the preconditioning intervention, participants rested again for 1
hour prior to a 5 min 45° limb elevation, immediately followed by a 30 min
tourniquet application (100 mmHg above resting systolic pressure) on the right
leg and a 2 hour period of reperfusion. The tourniquet pressure of 100 mmHg
above systolic was elected as it is sufficient to provide a bloodless field while
reducing the likelihood of associated negative side-effects (Worland et al., 1997).
Blood samples were obtained at Basal, immediately post intervention (PoI),
immediately pre-tourniquet application (PrT), 15 min post-tourniquet removal
(15PoT) and 120 min post tourniquet removal (120PoT) utilising the procedure
and controls outlined in section 3.4, with the addition of muscle samples collected
at PrT, 15PoT and 120PoT in accordance with section 3.5 (Figure 4.3).
60
3.7 Muscle Sample Preparation
Muscle samples were ground under liquid nitrogen prior to homogenisation (T10
Basic, IKA, Thermo Fisher Scientific, Loughborough, UK) on ice in 1 mL TRIzol
reagent followed by a 10 min incubation period on ice. RNA was extracted
utilising the method described in section 3.10.
3.8 Protein Carbonyl Quantification
PC is a widely accepted measure of protein oxidation (Powers et al., 2010b) and
has been cited as more stable marker of oxidised structural modifications in
Tourniquet Ischemia
Recovery Hypoxic Exposure Reperfusion
No preconditioning
TOR Condition
HYP Condition
SHAM Condition
Time (min)
14:55 11:30 12:30
0
13:10 14:10 14:40
0
16:55
Basal PoI PrT 15PoT 120PoT
Figure 3.3: Experimental design for all conditions. Blood samples (↑) were
obtained at Basal, PoI, PrT, 15PoT and 120PoT with additional
gastrocnemius tissue ( ) collected at PrT, 15PoT and 120PoT.
61
comparison to the more commonly used transient measures of MDA (Pantke et
al., 1999), therefore, this particular marker was chosen and analysed using
commercially available kits (Protein Carbonyl Colorimetric Assay Kit, 10005020,
Caymen Chemical Company Company, Michigan, USA).
Two hundred µL of pre-treated lithium heparinised plasma (section 3.4.3) was
added to 800 µL of 2,4-dinitrophenylhydrazine acting as the sample tube while
200 µL of plasma was added to 800 µL of 2.5 M hydrochloric acid to serve as the
control tube. All tubes were required to incubate in the dark for 1 hour at room
temperature with a brief vortex every 15 min. 1 mL of 20% trichloroacetic acid
(TCA) was added to each tube, briefly vortexed and incubated on ice for 5 min
prior to centrifugation at 10 000 G for 10 min at 4°C. This was followed by a 10%
TCA wash, incubation on ice for 5 min and centrifuged at 10 000 G for 10 min at
4°C. Supernatant was discarded and the pellet suspended in a 1:1 ethanol/ethyl
acetate wash before undergoing a thorough vortex and centrifugation at 10 000 G
for 10 min at 4°C. This was repeated twice more before the pellet being re-
suspended in 500 µL of guanidine hydrochloride and centrifuged at 10 000G for
10 min at 4°C. An aliquot of 220 µL of both sample and control was added to a
96-well plate and the absorbance was measured at 360 nm using a microplate
reader (Sunrise™, Tecan, Reading, UK). All samples and standards were analysed
in duplicate.
PC concentration was analysed using the subsequent equation:
( )
62
Corrected absorbance (CA) was produced through the subtraction of the average
control absorbance from the average sample absorbance with 500 µL/200 µL
providing the original sample concentration and 0.011 µM-1
as the actual
extinction coefficient for 2,4-dinitrophenylhydrazine at 370 nm. The intra and
inter-assay coefficient of variance are 4.7% and 8.5% respectively.
To assess the protein content of the sample, a 1:10 dilution of sample control to
gunadine hydrochloride was prepared, the absorbance was determined at 280 nm
using a Nanodrop 2000c (Thermo Fsiher Scientific, Loughborough, UK) and
calculated from a bovine serum albumin standard (0.25-2.0 mg∙mL-1
) curve using
the following equation:
( ) (
)
The final assessment of carbonyl content is produced via the subsequent equation:
( ) ( )
( )
3.9 Glutathione Analyses
Glutathione has been reported to provide a useful marker for disturbances to the
redox balance (Powers et al., 2010b). The most commonly used technique is high
performance liquid chromatography; however, this involves large quantities of
time pre- and post-assay procedure (Asensi et al., 1994) and can involve varying
degrees of GSH recovery (Ostman et al., 2004). Spectrophotometric techniques
are often utilised via verification of glutathione in the ‘recycling method’ of
Ellman’s reagent (5,5′-dithio-bis-2-nitrobenzoic acid (DTNB)) and GSH
63
measuring absorbance at 412 nm; providing convenience, sensitivity and accuracy
in various sample types (blood, urine, muscle, liver) (Rahman et al., 2006), thus
for these reasons, this method was chosen for this research project.
To determine the concentration of TGH previously treated blood (50 µL; section
3.4.3) was diluted to 1:40 with assay buffer solution and transferred to a 96-well
plate in accordance with the manufacturer’s instructions (Glutathione (Total)
Detection Kit, ADI-900-160, Enzo Life Sciences, Exeter, UK). A standard curve
was created through serially diluting 50 µL GSSG standard and 50 µL of assay
buffer solution (100-12.5 pmol). A 150 µL mixture of DTNB and 10 µL
glutathione reductase was added to all wells to produce 5-thio-2-nitrobenzoic acid
(TNB) which measured absorbance at 405 nm in a microplate reader (Sunrise™,
Tecan, Reading, UK) every minute for 10 min. For determination of GSSG, the
method outline above was replicated with the addition of samples first being
treated with 1 µL of 2M 4-Vinylpyridine (Sigma Aldrich, Dorset, UK) to block
any free thiols from cycling the reaction. 4 µL of 2M 4-Vinylpyridine was added
to 200 µL of GSSG standard to produce a standard curve. Samples and standards
were incubated for 1 hr and analyses were identical to the protocol for TGH.
Reduced glutathione was calculated via subtraction of GSSG concentrations from
TGH and a final GSH/GSSG ratio was computed. All standards and samples were
run in triplicate and an average was taken. The intra- and inter-coefficient of
variance for the assay kits was 3.4% and 3.6% respectively.
64
3.10 RNA extraction
RNA was extracted using previously validated methods (Chomczynski and
Sacchi, 1987). Briefly, chloroform (Sigma Aldrich, Dorset, UK) was added to
(200 µL for muscle samples; 40 µL for leukocytes samples) samples suspended in
TRIzol® reagent, then vortexed and left to incubate on ice for 10 min prior to
centrifugation at 17 000 G for 15 min at 4°C. The resulting sample separates into
an aqueous clear phase containing RNA and chloroform; a small white interphase
comprising of DNA and protein; and a large pink phase containing TRIzol and
cellular remnants. The aqueous phase was carefully aspirated into a fresh 1.5 mL
RNA-free eppendorf and equal volume of ice-cold propan-2-ol (Sigma Aldrich,
Dorset, UK) was added before a 15 min incubation period on ice and subsequent
centrifugation at 17 000 G for 15 in at 4°C. The supernatant was removed and the
sample was washed with ice-cold 75% ethanol (Sigma Aldrich, Dorset, UK) (1
mL for muscle samples; 100 µL for leukocytes samples) ahead of centrifugation at
5 400 G for 8 min at 4°C. Two additional ethanol washes were performed.
Remaining ethanol was aspirated and the pellet was allowed to air dry for 5 min
prior to the addition of 50 µL of RNA storage solution (Invitrogen, Paisley, UK),
followed by a final vortex for 90 s.
All procedures outlined above were performed using RNA-free pipette tips and
pipettes that were solely used for RNA work, on surfaces and equipment which
had been decontaminated with 70% industrial methylated spirit and RNase ZAP
(Ambion, The RNA Company Cheshire, UK) prior to commencement of RNA
65
work. Fresh tips were used for each sample to avoid sample cross-contamination.
The samples were frozen at -80°C for future RNA concentration quantification.
3.11 RNA concentration quantification
RNA concentrations and purity were calculated by spectrophotometry analysis
utilising the Nanodrop 2000c. 2 µL of RNA storage solution was placed onto the
pedestal as the blanking solution prior to sample measurement. Subsequently, 1µL
of sample was placed onto the pedestal and measured at wavelengths of 260 nm
and 280 nm. The ratio of 260/280 was used to assess the purity of the RNA
sample, where the ratio 2.0 was considered to be “pure” RNA. Samples were
considered “high quality” at ratios in the range of 1.90 – 2.10 and were accepted
for use in quantitative real-time polymerase chain reaction (RT-PCR).
Concentrations of RNA were calculated using a modification of the Beer-Lambert
equation:
( ) ( ) ( )
( )
Where c is the nucleic acid concentration; A is the absorbance; ε is the
wavelength-dependant extinction coefficient (40 ngcm·µL-1
for RNA); and b is
the pathlength.
3.12 One-step quantitative real-time polymerase chain reaction
RT-PCR was performed on a thermal cycler (RotorGene, Qiagen, Manchester,
UK) using QuantiFast® SYBR® Green RT-PCR kits (Qiagen, Manchester, UK)
containing: (2x) QuantiFast SYBR Green RT-PCR Master Mix (HotStarTaq®
66
plus DNA Polymerase, Quanitfast SYBR green RT-PCR buffer, dNTP mix
(dATP, dCTP, dGTP, dTTP), ROX passive reference dye) and (1x) QuanitFast
reverse transriptase (RT) mix (Omniscript® RT, Sensiscript® RT).
Primers in Table 3.2 were designed by Sigma Aldrich (Dorset, UK). 20 µL of
reaction mix (10 µL of SYBR green, 0.15 µL forward primer, 0.15 µL reverse
primer, 0.2 µL reverse transcriptase, 9.5 µL of sample (70 ng∙µL-1
of RNA)) was
distributed using an automated pipetting machine (QiAgility, Qiagen, Manchester,
UK).
The amplification program involved a preliminary denaturation phase at 50°C for
10 min followed by further holding at 95°C for 5 min. Samples then undertook 40
cycles of denaturation lasting 10 s at 95°C with a subsequent annealing and
extension phase for 30 s at 60°C. SYBR green fluorescence was measured after
each cycle. Melting curve analysis was then performed concluding the 40 cycles,
where samples were incubated at 50°C and heated to 99°C with the fluorescence
measured every 1°C increase. All samples were performed in duplicate.
67
Table 3.2: Primer Sequences
Target Gene Primer Sequence (5'-3'-)
Reference
Sequence
Number
Amplic
on
Length
(bp)
GC%
Content
B2
microglobuli
n
Forward:
CCGTGTGAACCATGTGAC
T
NM_0040
48 19 52.63
Reverse:
TGCGGCATCTTCAAACCT 18 50.00
HSP 72
Forward:
CGCAACGTGCTCATCTTT
GA
NM_0053
45 20 50.00
Reverse:
TCGCTTGTTCTGGCTGATG
T
20 50.00
HSP32
Forward:
CAGCAACAAAGTGCAAGA
T
NM_0021
33 19 42.11
Reverse:
CTGAGTGTAAGGACCCAT
C
19 52.63
3.12 Quantitative real-time polymerase chain reaction analyses
Samples displaying multiple peaks in the melting curve were excluded from
further analyses. RotorGene software plotted the sample fluorescence against
cycle number on a graph and the cycling threshold was manually positioned above
background fluorescence levels where there was an exponential rise in
fluorescence. Gene expression was determined through the ratio between the
target gene and the housekeeping gene, ß2-microglobulin, and was calculated
using the comparative threshold cycle (2-ΔΔCT
) method (outlined by Schmittgen
and Livak (2008)), where relative gene expression was determined using 2-ΔΔCT
.
68
3.13 Statistical Analyses
All data was analysed using the statistical software package IBM SPSS version
19.0 (SPSS Inc, Chicago IL, USA). Prior to any performance of inferential
statistics, descriptive tables and graphical methods (Q-Q plots and scatter plots)
were utilised to check for statistical assumptions. All data presented was deemed
to be normally distributed. A number of outliers were observed during exploratory
data analysis and were subsequently removed prior to performance of inferential
statistics (n = 6 in each condition unless otherwise stated). A one-way analysis of
variance (ANOVA) was used to assess for statistical differences between
participants’ anthropometric data. A one-way repeated measures ANOVA was
utilised to establish significant differences between haemoglobin saturation and
HR during the hypoxic intervention period. Sphericity was assumed for all
repeated measures analysis. Linear mixed models (LMMs) were used to identify
significant group x time interactions in the remaining dependant variables across
all groups. In the event of a significant F ratio for both LMMs and one-way
repeated measures ANOVAs, the post-hoc test Sidak was used to locate
significant pairs. LMMs were chosen as this particular type of statistical analyses
allows for missing data, for non-independent data and the best appropriate
covariant structure to be selected (Field, 2009). The most suitable covariant model
was decided using the difference in -2 restricted log likelihood figures and the
number of parameters of the two models tested against the χ2 critical statistic
(Field, 2009). Furthermore, residuals were checked for normality and
homogeneity of variance using Q-Q plots and scatter plots respectively, and were
considered plausible for all dependant variables. Statistical significance was
69
assumed at p < 0.05. Finally, Cohen’s effect sizes (ESs) for independent means
were calculated utilising the formula outlined by Cohen (1992):
The quantity d is the standardised mean difference, where µa and µb are separate
means. The value σ is the pooled standardiser and is computed using the
subsequent equation described by Olejnik and Algina (2000):
√( )
( )
( ) ( )
The quantities na, SDa and nb, SDb represent the sample size and SD for µa and µb
respectively. The ES was established as: small (d = 0.2), medium (d = 0.5) and
large (d = 0.8) effects (Cohen, 1992).
70
Chapter 4: Results
71
No significant differences (p ≥ 0.34) in participant demographics were observed
between SHAM, HYP or TOR, with the exception of age (F2,15 = 4.36, p = 0.32)
noted between conditions TOR and SHAM (p = 0.032) (Table 3.1).
A significant main effect displayed a decrease in haemoglobin saturation (F8,40 =
17.331, p < 0.001) between baseline and all subsequent time points (p < 0.05) in
the HPC intervention experienced by HYP. However, there was no significant
main effect (F8,40 = 1.130, p = 0.365) in HR noted by the same exposure (Figure
4.1).
0 5 10 15 20 25 30 35 40
50
60
70
80
90
70
75
80
85
90
95
100
Oxyhaemoglobin saturation
HR
*
** ** *
* *
Time (min)
HR
(b
ea
ts m
in-1
)
Ox
yh
aem
og
lob
in s
atu
ra
tio
n (
%)
Figure 4.1: Mean HR and oxyhaemoglobin saturation during
HYP intervention. * indicates significant difference v.s.
baseline value. Error bars represent SD. Abbreviations: HR –
heart rate
72
4.1 Circulatory stress and redox markers
There were no significant (p > 0.05) group x time interaction effects for leukocyte
Hsp72 (F = 1.195, p = 0.347), leukocyte Hsp32 (F = 1.406, p = 0.244), PC (F =
0.681, p = 0.707), TGH (F = 0.510, p = 0.844), GSSG (F = 0.510, p = 0.844),
GSH (F = 0.856, p = 0.564) or GSH/GSSG (F = 1.959, p = 0.105) (Table 4.1).
73
Table 4.1: Mean (SD) systemic circulatory stress and redox markers across basal, immediately post-intervention (PoI), immediately pre-
tourniquet application (PrT), 15 min post-tourniquet removal (15PoT) and 120 min post-tourniquet removal (120PoT)
Measure Basal PoI PrT 15PoT 120PoT
SHAM HYP TOR SHAM HYP TOR SHAM HYP TOR SHAM HYP TOR SHAM HYP TOR
Hsp72 (relative
fold change from
basal)
1.46
(0.42)
1.29
(0.39)
1.72
(0.54)
1.46
(0.55)
1.23
(0.44)
1.31
(0.17)
1.43
(0.41)
1.45
(0.36)
1.31
(0.35)
1.44
(0.43)
1.57
(0.31)
1.33
(0.14)
1.23
(0.44)
1.35
(0.27)
1.35
(0.21)
Hsp32 (relative
fold change from
basal)
1.34
(0.43)
1.08
(0.24)
1.47
(0.55)
1.09
(0.33)
1.14
(0.24)
1.23
(0.40)
0.86
(0.14)
1.10
(0.28)
1.29
(0.29)
0.86
(0.28)
1.24
(0.27)
1.17
(0.33)
1.04
(0.45)
1.02
(0.41)
1.08
(0.38)
Protein Carbonyl
(nmol·mL-1
)
0.56
(0.14)
0.56
(0.19)
0.69
(0.24)
0.54
(0.16)
0.42
(0.13)
0.61
(0.13)
0.64
(0.13)
0.58
(0.08)
0.69
(0.21)
0.65
(0.18)
0.60
(0.24)
0.56
(0.15)
0.65
(0.32)
0.69
(0.15)
0.63
(0.21)
Oxidised
glutathione
(pmol)
190
(87)
155
(70)
177
(96)
178
(70)
135
(34)
140
(35)
166
(52)
150
(63)
216
(127)
174
(50)
216
(56)
173
(69)
205
(83)
202
(75)
208
(97)
Reduced
glutathione
(pmol)
3815
(603)
4441
(598)
4081
(607)
3874
(653)
5218
(895)
4431
(520)
4021
(557)
4256
(998)
4397
(614)
4386
(366)
4163
(720)
4389
(864)
4224
(390)
4280
(585)
4353
(870)
Reduced/oxidised
glutathione ratio
23.0
(9.1)
31.4
(12.2)
22.7
(8.6)
24.6
(9.6)
29.3
(5.4)
28.9
(7.4)
27.1
(11.6)
23.9
(7.3)
18.6
(7.6)
27.6
(10.7)
20.5
(3.6)
23.1
(8.7)
23.0
(7.7)
22.4
(2.2)
20.5
(9.6)
Total glutathione
(pmol)
4006
(646)
4596
(650)
4258
(580)
4052
(649)
4352
(936)
4434
(561)
4187
(532)
4358
(888)
4596
(555)
4374
(557)
4343
(797)
4563
(828)
4395
(462)
4488
(580)
4625
(399)
Hsp – heat shock protein gene expression
74
4.2 Localised muscle stress markers
Hsp72 gene expression
Significant group x time interaction effects (F = 3.058, p = 0.048) were observed
in muscle Hsp72 relative gene expression. There was a 76% mean increase
between PrT and 15PoT (95% CI -3.771, -0.124; p = 0.035) in SHAM displaying
a large ES (1.44). Also, a pronounced 116% increase between PrT and 120PoT
(95% CI -3.779, -0.400; p = 0.014) was noted in TOR also producing a large ES
(1.59). Furthermore, there was a 51% and 50% decrease in HYP (95% CI 0.634,
3.934; p = 0.007) and TOR (95% CI 0.675, 4.114; p = 0.006) respectively when
compared to SHAM at 15PoT both demonstrating large ESs (1.90 and 2.19
respectively) (Figure 4.2). (SHAM, n = 5; HYP, n = 6; TOR, n = 6).
Figure 4.5: Hsp72 relative gene expression
PrT
15PoT
120P
oT
0
2
4
6
8SHAM
HYP
TOR
a
a,c,d
b
b
cd
Hsp
72
(rela
tiv
e f
old
ch
an
ge f
ro
m b
asa
l)
Figure 4.2: Mean muscle Hsp72 relative gene expression at
immediately pre-tourniquet application (PrT), 15 min post-
tourniquet removal (15PoT) and 120 min post-tourniquet
removal (120PoT) in all conditions. Like letters denote
significant differences (p < 0.05) between mean values. Error
bars represent SD.
75
Hsp32 gene expression
There were no significant group x time interactions (F = 0.147, p = 0.961) in
muscle Hsp32 gene expression over PrT, 15PoT and 120PoT (Figure 4.3)
(SHAM, n = 5; HYP, n = 6; TOR, n = 6).
Figure 4.6: Hsp32 relative gene expression
PrT
15PoT
120P
oT
0
1
2
3
4
5
SHAM
HYP
TOR
Hsp
32
(rela
tiv
e f
old
ch
an
ge f
ro
m b
asa
l)
Figure 4.3: Mean muscle Hsp32 relative gene expression at
immediately pre-tourniquet application (PrT), 15 min post-
tourniquet removal (15PoT) and 120 min post-tourniquet
removal (120PoT) in all conditions. No significant
interaction effects were noted at any time point Error bars
represent SD.
76
Chapter 5: Discussion
77
The purpose of the present study was to examine whether HPC and IPC elicited a
reduction in oxidative stress to knee surgery specific tourniquet application.
Furthermore, to elucidate a time-course for circulatory oxidative stress markers
(GSH, GSSG, GSH/GSGG ratio, PC); in addition to circulatory (leukocyte) and
localised (muscle) stress protein expression (Hsp72 and Hsp32). In contrary to the
hypothesis, the results revealed that a bout of either HPC or IPC did not produce a
statistically significant reduction in systemic Hsps, redox stress markers, or in
localised Hsp32 in comparison to control. However, both HPC and IPC did
demonstrate a significant reduction in Hsp72 at 15PoT in the localised
gastrocnemius tissue from TKR specific tourniquet application.
5.1 Circulatory redox and stress markers
Interestingly, the intervention groups did not display a significant difference in
oxidative stress markers in comparison to the control following TKR specific
tourniquet application. This is in disagreement with Koca et al. (2011) who
observed stable redox markers (malondialdehyde (MDA), total oxidant and
antioxidant capacity) in the IPC group in comparison to a significant negative
response noted in control following knee arthroscopy surgery. However, the
authors failed to implement any subject dietary restrictions, which could have
markedly affected the subjects’ antioxidant capacity (Powers et al., 2010b) and
could potentially have impacted upon the results published. Furthermore, smoking
has been shown to increase ROS production (Kalra et al., 1991), however, Koca
and colleagues (2011) did not exclude smokers from their research, thus the
experimental findings displayed may not be a true representation of their
78
experimental manipulation. Finally, the measure of MDA via thiobarbituric acid
assay utilised by Koca et al. (2011) is considered to be an unacceptable measure,
as the majority of thiobarbituric acid material found in the body is not related to
MDA (Powers et al., 2010b), perhaps explaining the differing redox results
between the current study.
It could be speculated that the 30 min bout of limb ischemia utilised here did not
induce a sufficient level of stress to observe the hypothesised potential favourable
effects bestowed from the preconditioning interventions in the systemic blood.
Previous surgical literature demonstrating the beneficial effects of IPC through
circulatory markers implemented a far greater ischemic periods (mean ± SD; 89 ±
9 min (Koca et al., 2011)). This extended ischemic period would induce greater
quantities of activated leukocytes and ROS into the circulation, thus stimulating
further cellular structure oxidation. Therefore, without the initial systemic
oxidative burst, preconditioning would not provide a noticeable benefit. Indeed,
this hypothesis is reinforced via the stable leukocyte stress response (Hsp) data
depicted in Table 4.1.
HSPs are up-regulated during a variety stressors, such as oxidative stress, hypoxia
and ischemia (Morton et al., 2009). The homeostatic insults lead to protein
denaturation and unfolding, initiating the heat shock response, thus permitting
heat shock factor 1 (HSF1) to oligermerise and bind to the heat shock element
promoting gene transcription (Noble et al., 2008). HSP72 provides cytoprotection
through refolding of denatured proteins and rescuing apoptotic cells via
interruption of the programmed death cascade (Taylor et al., 2011). An increase in
79
Hsp72 would demonstrate greater cellular stress (Theodorakis et al., 1999),
however, as aforementioned; no significant increases in leukocyte Hsp72 were
noted in peripheral blood in any of the conditions following the TKR specific
tourniquet application (Table 4.1). Therefore, it could be inferred that there was
no systemic stress following 30 min limb ischemia.
Surprisingly, there were also no significant changes in leukocyte Hsp72
concentration at PoI in HYP or TOR following the preconditioning (Table 4.1). A
rise in Hsp72 following HPC or IPC would be expected as the initiation of both
interventions invokes an oxidative burst (Konstantinov et al., 2004; Taylor et al.,
2010), thus stimulating the heat shock response. In contrast to the results in the
current study, Konstantinov et al. (2004) showed an increase in peripheral
leukocyte Hsp72 using microarray analyses following IPC in the forearm.
Although the authors observed a rise in leukocyte Hsp72, there is evidence to
suggest that microarray analyses can overestimate gene expression (Feldman et
al., 2002). Furthermore, Taylor et al. (2010) noted a significant increase in Hsp72
following an acute hypoxic exposure, which again is in contrary to the data
following HPC (Table 4.1). The disparity between Taylor et al. (2010) and the
present study may be due to the longer hypoxic period utilised by the authors, thus
providing greater systemic stress to induce a larger up-regulation in Hsp72.
Both Taylor et al. (2010) and Konstantinov et al. (2004) used different techniques
(flowcytometry and microarray respectively) of quantifying the changes in stress
protein response in comparison to the present study (RT-PCR). However,
measurement of gene expression via RT-PCR is considered to be a sensitive and
80
reproducible method in which to quantify gene expression (Wong and Medrano,
2005), as such, would be able to identify the appearance of small changes in gene
expression.
A rise in leukocyte Hsp32 would be anticipated following an oxidative stress
insult (Fehrenbach et al., 2003). The rapid induction of HSP32 has been proposed
to stimulate protection via the catabolism of the reactive free heme into carbon
monoxide and biliverdin (Gozzelino et al., 2010). However, the present study did
not observe a significant increase in leukocyte Hsp32 (Table 4.1). Despite the
evidence depicting the vital role HSP32 plays in counteracting oxidative stress
(Gozzelino et al., 2010), no previous research has assessed Hsp32 with regards to
human limb ischemia in-vivo. Therefore, this finding (stable systemic Hsp32
concentrations following preconditioning and limb ischemia) can be considered
novel.
Glutathione concentrations have been previously measured in the systemic blood
following TKR surgery (Mathru et al., 1996; Karg et al., 1997). Interestingly, both
Karg et al. (1997) and Mathru et al. (1996) only observed changes in GSSG
immediately following reperfusion in the localised blood supply rather than the
systemic circulation. The observation of stable systemic glutathione markers is in
accordance with the present study’s findings (Table 4.1). This would suggest that
the localised circulation offered protection to the remote blood supply from
potentially damaging ROS. Indeed, it has been shown that intact erythrocytes
scavenge H2O2, providing vital protection to distant organs (Toth et al., 1984).
The non-significant change in systemic glutathione markers also demonstrates the
81
safe duration of at least 30 min limb occlusion, as previous research has
associated prolonged limbed ischemia (3 hrs) with multiple organ dysfunction
syndrome via circulating ROS and activated leukocytes (Yassin et al., 2002).
The assessment of PC concentration in the blood following knee specific
tourniquet mediated ischemia is a novel finding. In fact, the majority of studies
assessing limb occlusion have evaluated PC concentrations in the localised tissue
(Ozyurt et al., 2006; Avci et al., 2012; Ozkan et al., 2012). In contrast to the data
provided here, previous studies assessing plasma PC concentrations following
ischemia-reperfusion in other tissues (cardiac, intestinal) displayed a significant
increase in plasma oxidised protein concentrations (Narayani et al., 2003). It could
be speculated that different sampling sites (i.e. systemically or directly from the
ischemic site) and varying experimental durations of ischemia-reperfusion utilised
by Narayani et al. (2003) could account for the disparity in plasma PC
concentrations in comparison to the current study. However, the authors failed to
state this information, creating difficulties in producing valid comparisons to the
work presented here.
Furthermore, the stable concentrations of PC observed here (Table 4.1) are not
surprising considering the lack of significant variation in the GSSG
concentrations. Indeed, the simultaneous increase in both GSSG and PC
concentrations has previously been noted following ischemia reperfusion in rat
hindlimb (Grisotto et al., 2000). The ischemic bout promotes the formation of
ROS (such as OH•, O2
-) and ROS associated intermediates (H2O2) upon
reperfusion, inducing cellular protein oxidation. GSH metabolises the ischemic
82
mediated H2O2, thus removing the intermediate in the chain reaction that
synthesises the extremely reactive OH•, invariably forming GSSG and minimising
protein damage (Mari et al., 2013). Thus, a fairly constant GSH/GSSG ratio
indicates minimal disruption to the redox balance (Asensi et al., 1999), therefore,
negligible protein oxidation would occur.
5.2 Muscle HSP expression
The blunting of the tourniquet induced response noted by the reduction in Hsp72
displayed in both HYP and TOR in comparison to SHAM at 15PoT, also showed
a large effect size (1.90 and 2.19, respectively) (Figure 4.2). Similar observations
were noted by Bushell et al. (2002). The authors showed IPC did not stimulate an
increase in skeletal muscle Hsp72, when a dramatic rise was observed in the
control condition. Interestingly, the authors still witnessed protection in the IPC
tissue following an ischemic insult, in spite of the stable Hsp72 concentrations,
thus, indicating that perhaps HSP72 does not play a role in early preconditioning.
Therefore, the blunted response observed in both TOR and HYP in comparison to
SHAM would suggest a reduction in cellular stress from conditioning
(Theodorakis et al., 1999) rather than an IPC mechanism.
IPC acts in a biphasic pattern, with the early phase of protection being initiated
immediately post preconditioning and lasting up to 3 hrs (Yang et al., 2010);
while the delayed phase occurs 24 hrs following the stimulus, enduring for up to
72 hrs (Hausenloy and Yellon, 2010). Previous research implicates adenosine as a
key molecule in initiating early IPC (Liu et al., 1991). The ischemic environment
leads to the degradation of AMP via 5’-nucleotidase (Kitakaze et al., 1995),
83
directly stimulating protein kinase C (PKC) (Carden and Granger, 2000). PKC has
been proposed to act upon the mitochondrial potassium ATP (mKATP) channel and
the mitochondrial permeability transition pore (mPTP), with the former inducing
an influx of K+, further stimulating PKC (Sadat, 2009). The activation of PKC
inhibits the mPTP, whilst also activating cytochrome C oxidase, further increasing
cellular respiration, thus protecting the cell from excessive ROS (Sadat, 2009).
Besides adenosine, bradykinin can also stimulate PKC indirectly through ERK
and redox signalling, ultimately inducing PKC activation via ROS stimulation
(Cohen et al., 2007).
Although the mechanism described above explains the diminished tourniquet
mediated stress in TOR, it does not directly provide evidence for the same Hsp72
decrease noted in HYP. However, it has been previously stated that HPC acts
through similar redox mechanisms as IPC (Zuo et al., 2013). In fact, this would be
logical considering the increase in adenosine is produced via the degradation of
AMP (Kitakaze et al., 1995) that has accumulated through the disturbance of
aerobic respiration (Jennings and Reimer, 1991), which can be produced by both
hypoxia and ischemia. This has been confirmed via pharmaceutical blockade of
PKC, the mKATP channel and adenosine receptors, ultimately abolishing the
protective effects of HPC in cardiomyocytes (Nojiri et al., 1999). Interestingly,
the same study noted that nicorandil, a mKATP channel opener, which has
previously been shown to induce HPC protection from IRI, did not bestow the
same protection when PKC was inhibited, demonstrating that mKATP channel
opening leads to PKC activation, which is in contrast to the activation mechanism
of IPC. Therefore, the reduction in ischemic mediated stress observed in both
84
TOR and HYP in the present study (Figure 4.2) could be produced via the
aforementioned mechanism.
A significant increase in Hsp72 at 120PoT compared to PrT was noted in TOR
(Figure 4.2). Although not in skeletal muscle, a similar rise in Hsp72 was cited in
rabbit cardiac tissue 3 hrs following IPC (Tanaka et al., 1998). Previous research
has described HSP72 as a major instigator in producing the delayed effects of IPC
to afford protection for future insults (Lepore et al., 2000; Okubo et al., 2001; Li
et al., 2003). Consequently, the sharp rise in Hsp72 at 120PoT displayed by TOR
could potentially be explained by the delayed phase of IPC. However, the
elevation in Hsp72 noted here is following the combination of both IPC and TKR
specific tourniquet application, not just following IPC as mentioned by previous
research (Tanaka et al., 1998; Li et al., 2003), hence it is not classical delayed
IPC. Thus, it could be suggested that the rise in Hsp72 could merely be a result of
oxidative stress from the TKR specific tourniquet application (Lepore et al.,
2001); nonetheless if this were the case, a similar increase would have been
expected in SHAM (Figure 4.2). Therefore, it could be inferred that the presence
of increased Hsp72 concentrations could be explained by the delayed
preconditioning phenomenon from the IPC.
The postponed protection afforded by IPC has been attributed to de novo synthesis
of cytoprotective proteins such as HSPs, eNOS, cyclooxygenase-2 and MnSOD
(Hausenloy and Yellon, 2010). The up-regulation of these proteins occurs through
the activation of a multitude of transcription factors, stimulated by endogenous
mediators including PKC, adenosine and tyrosine kinase (Heusch et al., 2008; Yin
85
et al., 2009). The synthesis of HSP70 is speculated to refold sub-lethal damaged
proteins or diminish their interactions with viable proteins during the prolonged
ischemia/reperfusion bout (Marber et al., 1993). This is further supported by
evidence of infarct reduction in rabbit models overexpressing HSP72 (Okubo et
al., 2001). In addition to providing cytoprotection, HSP70 has also been shown to
mediate κ-opioid receptor stimulation, which are in part responsible for delayed
IPC (Zhou et al., 2001). This may partly explain the increase in Hsp72 at 120PoT
depicted in Figure 4.2.
In contrary to the research that considers HSP72 crucial to delayed IPC, it has also
been proposed that the occurrence of HSP72 24 hrs following IPC may in fact be
a marker of delayed preconditioning, rather than the mechanism involved in
providing protection (Pagliaro et al., 2001). Qian et al. (1999) demonstrated a
marked increase in HSP72 24 hrs following IPC in rat myocardium, however, this
rise did not induce protection in myocardial tissue. The authors concluded that the
discrepancy could be a variation in species response to delayed preconditioning.
Nevertheless, it has been proposed that PKC may also play a pivotal role in both
phases of preconditioning, with the activation of PKC in the delayed phase
governed by tyrosine kinase and NO, ultimately inducing further opening of the
mKATP channel (Pagliaro et al., 2001). However, recent novel research has
instigated the small non-coding microRNAs as another potential mechanism of
delayed IPC (Yin et al., 2009). Yin and colleagues (2009) demonstrated that
injection of microRNAs reduced the infarct size in murine hearts and up-regulated
HSP70, eNOS and hypoxia induced factor -1α, possibly through post-
transcriptional regulation of injurious genes. The rise in Hsp72 noted in the
86
current research (Figure 4.2) could potentially be a precursor to delayed
preconditioning; however the literature remains controversial regarding this
hypothesis.
Surprisingly, HYP did not display the dramatic rise in Hsp72 demonstrated by
TOR at 120PoT. This is unexpected considering the evidence outlined above
depicts that both HPC and IPC appear to share similar molecular mechanisms.
However, it could be hypothesised that HYP demonstrated a severe reduction in
oxidative stress during the TKR specific tourniquet application compared to TOR,
thus leading to reduced HSP response via hypoxia induced Hsp72 down-
regulation (Oehler et al., 2000). Furthermore, the negligible elevation in Hsp72 at
120PoT would also indicate the absence of HSP72 in inducing delayed
preconditioning as aforementioned in HYP. This is in disagreement with previous
literature, which has attributed HSP72 in producing the delayed preconditioning
phase (Engelman et al., 1995). Nevertheless, the study by Engelman et al. (1995)
displayed a rise in Hsp70 4 hrs following a prolonged hypoxic exposure post
HPC, thus it could be speculated that the stable Hsp72 demonstrated here (Figure
4.2) was merely a disparity in sampling time and may occur later than IPC.
Interestingly, the same study did not find an increase in Hsp70 ensuing HPC prior
to the sustained hypoxic insult. This is in line with the current study where no
significant difference was observed between HYP and SHAM at PrT. It could be
suggested that the absence of an increase in Hsp72 following HPC could
potentially be via the inhibition of the kinase, mammalian target of rapamycin
(mTOR). mTOR has been cited as a major kinase involved with crucial
phosphorylation of HSF1 following dissociation from HSPs (Chou et al., 2012).
87
However, acute normobaric hypoxia has been demonstrated to inhibit mTOR
function (D'Hulst et al., 2013), thus preventing HSF1 phosphorylation and further
transcription of Hsp72. This mechanism may explain the stable concentrations of
Hsp72 (Figure 4.2) observed in the current research.
The present study did not show a change in muscle Hsp32 between any of the
conditions (Figure 4.3). This is in disagreement with previous research showing
that Hsp32 expression transiently increased following HPC (Berger et al., 2010)
and greater protein expression succeeding IPC (Badhwar et al., 2004). It is
postulated that the lack of variation in Hsp32 concentrations could be via Hsp32
transcriptional repression during the hypoxic insult. Nakayama et al. (2000)
established that heme oxygenase-1 (HSP32) is down-regulated in ECs during the
hypoxic exposure. It has been postulated that the reduction in Hsp32
concentrations could consequently reduce the large energy expenditure associated
with heme degradation, preserving vital ATP stores (Nakayama et al., 2000).
Additionally, the reduction in translation of de novo HSP32 during the hypoxic
period would diminish the immediate production of CO, preventing its binding to
oxygen sensing heme molecules, invariably disrupting their function (Shibahara,
2003). Indeed, the transcription factor Bach-1 has been identified as a regulator of
HSP32 (Ogawa et al., 2002). The presence of heme negatively affects Bach-1’s
ability to repress HSP32 gene expression via DNA binding (Ogawa et al., 2002).
The present data did not show a significant down-regulation of muscle Hsp32
throughout the assessed time-points, nevertheless, this could possibly be due to
the short hypoxic exposure (40 min hypoxia, 60 min recovery, 30 min ischemia)
88
experienced by the participants, thus resulting in the equalised Hsp32
concentrations observed over the experimental period (Figure 4.3). It could also
be speculated that the stable concentrations of Hsp32 may be a fibre type specific
response. Type I muscle fibres have been shown to readily express HSP32 in
comparison to a blunted response noted in type II fibres (Vesely et al., 1999). The
lateral head of the gastrocnemius consists of equal proportions of both fibre types
(Edgerton et al., 1975), therefore the response observed in the present study may
only be proportional to the percentage of type II fibres in the muscle.
5.3 Application of results
Recently, the use of IPC as non-pharmaceutical, non-invasive intervention has
been cited to diminish postoperative pain following TKR surgery (Memtsoudis et
al., 2010). Although the difference in pain noted by Memtsoudis and colleagues
(2010) was not the main aim of the study, the simplicity of IPC allowed it to be
performed during draping/surgical preparation ensuring no delay to
commencement of surgery, thus, even a modest improvement in patient perceived
pain should be seen as a positive and an advocate for the use of IPC as an addition
during TKR surgery. The present study has also demonstrated that HPC provides
similar protective effects to IPC against 30 min tourniquet mediated ischemia. In
addition, the simplicity of the HPC protocol used here could also be undertaken
during the pre-operative phase while the patient is still on the ward. The present
HPC protocol has the added benefit of less staff involvement than IPC through
redundancy of tourniquet inflation/deflation monitoring.
89
Furthermore, length of stay has been demonstrated to significantly decrease
following IPC in comparison to control in TKR out-patients (Memtsoudis et al.,
2010). Although these results (reduction in pain and LOS) are promising, the
study itself was not specifically designed to identify clinical outcomes; therefore
further research in this area is warranted. The similar effects induced by protection
bestowed from HPC and IPC in the current study could potentially reduce length
of stay comparatively to Memtsoudis et al. (2010) through attenuation of muscular
tissue damage mediated through tourniquet use. Thus, if this were confirmed in a
study designed to solely address this entity, it could potentially have a large effect
upon clinical practise in the future.
5.4 Limitations
Firstly, many antioxidants are procured naturally from the diet, all with varying
half-lives, potentially influencing the antioxidant defence capacity of the
individual (Powers et al., 2010a). Although every effort was made to minimise
this (standardised evening, morning and afternoon meal), it is extremely
challenging to control participants’ diet over a long period of time, while ensuring
continued participation. Therefore, the lack of change in oxidative stress markers
could have partly been due to dietary variation.
Secondly, difficulties obtaining blood samples from the site of ischemia (i.e. the
right calf) hindered the possibility of further inferences from this data. If samples
were collected from the ischemic site, the data may have alluded to a greater
insight into the potential link between the localised tissue and the systemic blood.
Previous research (Karg et al., 1997; Garcia-de-la-Asuncion et al., 2012) has
90
shown disparity between markers at the systemic and localised sites, consequently
the oxidative stress markers measured in the present study may not depict the full
cascade of events.
Finally, the duration of limb ischemia utilised in this study (30 min) is lower than
commonly used during TKR surgery (mean ± SD; 79.9 ± 12.7 min (Chang et al.,
2012)). Therefore, it is expected that the oxidative stress would be greater during
surgery thus the current model performed in the study is not completely
ecologically valid.
5.5 Conclusions
Overall, systemic markers of oxidative stress did not change during the trial in any
condition, thus producing a stable time course for redox (GSH, GSSG, TGH,
GSH/GSSG, PC) and stress protein markers (Hsp72, Hsp32) following both
preconditioning and TKR specific tourniquet application. In addition, HPC and
IPC did not induce a marked reduction in the systemic oxidative stress measures
compared with a control condition. However, the significant reduction in localised
cellular stress noted in both TOR and HYP at 15PoT is encouraging. This
provides further evidence for protection offered via IPC but also demonstrates the
potential of HPC in diminishing cellular stress associated with TKR specific
tourniquet application, although the precise mechanisms of action were not
alluded to in this study. The diminished localised stress provides a rationale for
the extension of this research into a clinical population to establish more
clinically-relevant qualitative measures for patient’s perceived pain and levels of
surgical success.
91
Finally, to conclude this thesis, it is appropriate to revisit the experimental aims
described at the end of the literature review section. The aims are restated below
and an appropriate answer has been provided.
1) Quantify the time course for redox disturbances to the systemic and
localised circulation via analysis of PC, GSH, GSSG and TGH, following
hypoxic and ischemic preconditioning, in addition to 15 min and 2 hrs
succeeding tourniquet mediated ischemia.
- Systemic circulation concentrations of PC, GSH, GSSG and TGH did not
significantly fluctuate from basal following any of the interventions
(SHAM, HPC, and IPC) and TKR specific tourniquet application.
Localised circulatory redox markers were not assessed due to problems
with localised blood collection.
2) Examine the time course for changes in Hsp72 and Hsp32 in localised
skeletal muscle, in addition to localised and systemic leukocytes utilising
the same time points as outlined in 1).
- HPC and IPC displayed a blunted Hsp72 response in skeletal muscle in
comparison to SHAM 15 min following tourniquet release. Additionally,
IPC displayed sharp increase 2 hrs post tourniquet release. Tissue Hsp32
and Systemic circulatory Hsp72 and Hsp32 did not show any alterations
following tourniquet release.
3) Evaluate the efficacy of both whole-body HPC and limb IPC based on the
observed changes in 1) and 2) from TKR specific tourniquet application.
- HPC and IPC displayed a blunted Hsp72 response in comparison to
SHAM 15 min following tourniquet release in skeletal muscle. Therefore,
92
suggesting that both HPC and IPC provided cellular protection to
tourniquet mediated oxidative in localised tissue.
5.6 Recommendation for future research
Completion of this thesis has generated several potential areas for future research,
specifically, establishment of oxidative stress markers and cell viability in the
localised muscle. Furthermore, the potential benefits of preconditioning should be
confirmed within a small clinical population undergoing TKR to establish more
clinically relevant measures (i.e. length of stay, Oxford Knee Score, patient
perceived levels of pain). The following section will provide a brief summary of
studies addressing these issues.
5.6.1 Determination of redox disturbance and cellular structure in muscle
tissue following HPC in a TKR specific tourniquet application
The confirmation of reduced cellular structural damage and minimised
disturbance to muscle redox balance would provide empirical evidence to the
effectiveness of HPC. In addition to the markers utilised here (GSH/GSSG, PC)
antioxidant enzymes (e.g. MnSOD) and assessment of cell viability would provide
a greater representation of the biochemical events occurring in the localised tissue
following tourniquet application. An additional muscle biopsy would be added at
24 hrs post tourniquet removal to assess changes peak changes in MnSOD levels
(Hoshida et al., 1993). Venous blood would also be obtained at biopsy sampling
times from the femoral vein to measure redox disturbances and ROS via spin
trapping (Villamena and Zweier, 2004) (Figure 5.1). The use of western blot
93
analysis and ELISA assay kits would confirm protein and thiol concentrations
respectively.
Figure 5.1: Schematic of proposed experimental design. The shaded and blocked
areas represent HPC and tourniquet ischemia respectively.
5.6.2 The feasibility and clinical relevance of HPC in TKR surgery - A small
clinical trial
The research would be performed in hospital and adhere to a standard operative
day. Participants would be screened for inclusion criteria (bleeding disorders,
immunocompromised, respiratory disorders etc.) (Memtsoudis et al. 2013) and
undergo the HPC intervention while on the ward before admittance to surgery. In
addition, pain (Visual analogue scale), analgesia consumption and muscular blood
oxygenation would also be assessed at baseline, 6, 24 and 48 hrs post-operation
(Figure 5.2). Assessment of standard TKR success criteria (Oxford Knee Scores,
physiotherapy milestones) would also be recorded. These data would test the
feasibility of utilising HPC in a hospital environment and provide clinically
relevant information to the effectiveness of the preconditioning succeeding
surgery.
Muscle biopsy
Venous blood
Muscle biopsy
Venous blood
Muscle biopsy
Venous blood
Muscle biopsy
Venous blood
Basal Post
- +2 hrs +24 hrs
94
Figure 5.2: Experimental design for the clinical trial. Shaded and blocked areas
represent HPC and TKR surgery respectively. Upon admittance variables would
be obtained and patients would be prepared for surgery prior to commencing
HPC. Succeeding surgery all measures would be collected while the patient is on
the ward.
Basal +6 hrs +24 hrs +48 hrs
Pain (visual analogue scale)
Analgesic consumption
Muscular blood oxygenation
95
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96
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Appendices
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Appendix A
Abstinence Criteria
Participant ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Alchol (72 hours)
Caffeine (72 hours)
Hot bath/sauna (48 hours)
Antioxidants (30 days)
Beta Alanine (15 weeks)
Glutathione (4 weeks)
Creatine (30 days)
Residing at Altitude (3 months)
Hyperthermic environments (3 months)
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Appendix B
Information Sheet
Department of Sport and Exercise Sciences
Bedford Campus
Polhill Avenue
Bedford
MK41 9EA
Dear Participant,
Thank you for showing an interest in participating in this novel research.
Please read this information sheet carefully before deciding whether to participate. If
you volunteer we thank you for your participation. If you decide not to take part there
will be no disadvantage to you of any kind and we thank you for considering the
request.
What is the aim of the project?
The purpose of the study is to establish whether a hypoxic (low O2 levels) exposure
prior to knee surgery specific tourniquet application, provides protection against tissue
damage within the subjected leg.
What type of participant is required?
Participants must be 18-35 years of age, male, and in good health.
What will the participant be required to do?
Participants will be required to attend the Sport Science Laboratories at the University
of Bedfordshire’s Polhill Campus on 2 occasions. You will be required to wear shorts,
and abstain from alcohol and caffeine for 72 hours prior to testing. In addition you will
be required abstain from intensive exercise and thermal events (sauna/very hot baths
(normal bath/shower temperatures are acceptable)). Further you will be required to
abstain from antioxidants, beta-alanine, creatine and glutamine for 30 days, 15 weeks,
30 days and 4 weeks respectively. Finally, please ensure you have not resided at altitude
or hot environments for the previous 3 months. On the day of testing, please consume
500 mL of water 1 hr prior to start of testing to ensure adequate hydration.
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Prior to testing you should complete a medical questionnaire, a blood analysis
participant screening form and informed consent form, to demonstrate that you are
physically capable of participating and that you understand what you are taking part in
and the reasons why.
Baseline Testing
Prior to experimental testing you will visit the University of Bedfordshire’s laboratories
once for baseline testing. This visit will require you to have your blood pressure, height,
weight and body composition measured via use of the bodpod for the later, which will
last around 30-40 minutes. Following this you will be allocated either to control, sham,
hypoxic, or ischaemic conditions. Although subjects will not be informed which
condition they have been placed in.
Testing Overview
Control condition:
Seated in a laboratory room for 40 mins.
Sham Condition:
Seated in a laboratory inhaling normal ambient air (20.9% O2) through a mask attached
to the Hypoxicator for 40 mins
Hypoxic Condition:
Seated in a laboratory inhaling hypoxic air (14.5% O2) through a mask attached to the
Hypoxicator for 40 mins.
Ischaemic Condition:
Seated in a laboratory and have a tourniquet placed around their non-dominant thigh.
Followed by 4 compression and release cycles lasting 5 minutes each. The tourniquet
will be inflated to 100 mmHg above resting systolic blood pressure.
Experimental Trial
All subjects will be required to attend the laboratories at 11:00 am on their day of
testing. Upon arrival you will be required to provide a urinal sample to assess hydration
status. If dehydrated, subjects will be asked to drink 500 ml of water. Participants will
then be asked to sit on a massage couch, during which time, two cannulae will be placed
in the antecubital region (lower arm) and the small sephinous vein (calf) by either a
suitably trained affiliate of the University of Bedfordshire or experienced medical
practitioner from Milton Keynes General Hospital (MKGH). Subjects will then be
requested to sit and rest for 1 hour. A blood sample from both cannulae will be obtained
to assess levels of fold change in heat shock protein (HSP) 72 and 32, in addition to
determining protein and oxidative stress concentrations.
Participants will then place, on to themselves, a heart rate (HR) monitor and an
oximeter (on to the great toe of the non-dominant leg). Subjects will then undertake
their allocated intervention as outline above. During this time HR and oxygen saturation
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(OS) will be monitored. On completion, blood samples will be taken from both
cannulae. Subjects will then be asked to sit and rest for 1 hour.
Next you will be asked lay in a supine (on your back) position, and provide a muscle
sample obtained from the lateral side of the gastrocnemius (calf), which will be obtained
by an experienced medical practitioner from MKGH. In addition to muscle samples,
blood will also be extracted from both cannulae at this point. A tourniquet will then
placed upon your non-dominant leg and inflated to 100 mmHg above resting systolic
pressure for 45 minutes. During this time your HR and OS will be measured. In addition
to these, your thermal sensation (how hot/cold you feel) and pain (via a visual analogue
pain scale) will be taken in consideration to your whole body and subjected leg. Once
the elected time has elapsed, the tourniquet and oximeter will be removed and another
muscle and blood sample will be obtained 15 min post removal of the tourniquet from
the same regions as previously stated.
Subjects will be required to return to have further blood and muscle samples taken 2
hours post removal of the tourniquet. Upon completion HR monitor will be removed.
What are the possible risks of taking part in the study?
There will be a trained first aider in the immediate vicinity throughout all testing.
During the muscle biopsy and blood sampling there is a slight risk of infection and you
may experience a degree of discomfort. However, the risk of infection will be kept to a
minimum through use of a designated clinical area and performed using sterile
techniques. The level of discomfort during blood sampling will be minimised through
the use of a trained phlebotomist (individual who takes blood samples) and during the
muscle biopsy an orthopaedic surgeon will be performing the procedure.
There is a potential risk of altitude sickness when exposed to oxygen levels of 14.5%.
This risk is minimum and all researchers involved are aware of the symptoms. During
the experimental trial you may experience some discomfort from the tourniquet,
although once removed, this will subside.
What if you decide you want to withdraw from the project?
If, at any stage you wish to leave the project, then you can without given explanation.
There will be no disadvantage to yourself should you wish to withdraw.
What will happen to the data and information collected?
Everyone that takes part in the study will receive their own results for the tests that they
complete for your own personal development and understanding. All information and
results collected will be remain anonymous and held securely at the University of
Bedfordshire and will only be accessible by the project team. Results of this project may
be published, but any data included will in no way be linked to any specific participant.
Your anonymity will be preserved.
What are the potential benefits of the study?
The present study will determine whether hypoxic and ischaemic interventions reduce
the amount of tissue damage sustained after tourniquet application. Consequentially, if
128
successful, may reduce time needed for rehabilitation inducing reductions in cost for
hospitals and clinics. In addition participants will obtain a full body composition profile
(% fat and lean mass) worth £50.
Questions are always welcome and you should feel free to ask either myself, James
Barrington, Dr Lee Taylor or an independent contact, Professor Angus Duncan at
anytime. See details below for specific contact details.
If you are interested in taking part in the project and would like to receive more details
about the studies please send an email to either:
James Barrington: 07734821427 Dr Lee Taylor
Email: [email protected] Email:[email protected]
Department of Sport and Exercise Sciences, Professor Angus Duncan
University of Bedfordshire Email:[email protected]
Bedford Campus,
Polhill Avenue, Bedford
129
Appendix C
CONSENT FORM
TO BE COMPLETED BY PARTICIPANT
NAME:…………………………………………………(Participant)
I have read the Information Sheet concerning this project and understand what it is
about. All my further questions have been answered to my satisfaction. I understand that
I am free to request further information at any stage.
I know that:
- My participation in the project is entirely voluntary and I am free to
withdraw from the project at any time without disadvantage or prejudice.
- I will be required to attend testing in the sport and exercise science laboratories
on 2 separate occasions to complete the project.
- As part of the study I will have to:
Undergo body composition measurements through use of the bodpod.
Have blood pressure measured.
Provide a urine sample for testing pretesting.
Have a cunnula placed in both antecubital region (inside of the arm) and in the
small saphenous vein (back of the calf).
Have heart rate monitored throughout testing.
Have oxygen saturation levels measured throughout testing.
Be exposed to one of the four following interventions:
Normoxic environment (20.9% O2, 775 mmHg) at rest for 40 mins.
Hypoxic environment (14.5% O2, 775 mmHg) at rest for 40 mins.
4 cycles of 5 mins compression and 5 mins reperfusion from a tourniquet
inflated to a 100 mmHg above resting systolic pressure placed on the
thigh.
Control environment – seated in the laboratory at rest for 40 mins.
Undergo 45 mins of tourniquet compression inflated to a 100 mmHg above
resting systolic pressure placed on the thigh.
Give an indication of pain and ratings of thermal sensation throughout testing.
Provide 10 blood samples and 3 skeletal muscle biopsies.
- I am aware of any risks that may be involved with the project.
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- All information and data collected will be held securely at the University
indefinitely. The results of the study may be published but my anonymity will be
preserved.
Signed:………………………………… (Participant) Date: ………………
131
Appendix D
BLOOD ANALYSIS
Please read the following:
a. Are you suffering from any known active, serious infection?
b. Have you had jaundice within the previous year?
c. Have you ever had any form of hepatitis?
d. Have you any reason to think you may be HIV positive?
e. Have you ever been involved in intravenous drug use?
f. Are you a haemophiliac?
g. Is there any other reason you are aware of why taking blood might be
hazardous to your health?
h. Is there any other reason you are aware of why taking your blood might be
hazardous to the health of the technician?
Can you answer Yes to any of questions a-g? Please tick your response in the box
below:
Yes No
Small samples of your blood (from finger or earlobe) will be taken in the manner
outlined to you by the qualified laboratory technician. All relevant safety procedures
will be strictly adhered to during all testing procedures (as specified in the Risk
Assessment document available for inspection in the laboratory).
I declare that this information is correct, and is for the sole purpose of giving the
tester guidance as to my suitability for the test.
Name ………………………………………
Signed ………………………………………
Date ………………………………………
If there is any change in the circumstances outlined above, it is your responsibility to
tell the person administering the test immediately.
The completed Medical Questionnaire (Par Q) and this Blood Sampling Form will be
held in a locked filing cabinet in the Department of Sport and Exercise Science
132
laboratories at the University for a period of one-three years. After that time all
documentation will be destroyed by shredding.
133
Appendix E
General pre-test medical questionnaire
To be completed by all subjects before participating in practical sessions.
Name: ………………………………………………….
Age:…………… Gender: M / F
1 Are you in good health? Yes / No
If no, please explain:
2 Are you pregnant or have you given birth in the last 6 months? Yes / No
3 How would you describe your present level of moderate activity?
< once per month
once per month
2-3 times per week
4-5 times per week
> 5 times per week
4 Have you suffered from a serious illness or accident? Yes / No
If yes, please give particulars:
5 Are you recovering from an illness or operation? Yes / No
If yes, please give particulars:
6 Do you suffer, or have you ever suffered from:
Respiratory conditions (asthma, bronchitis, tuberculosis, other)? Yes / No
Diabetes? Yes / No
Epilepsy? Yes / No
High blood pressure? Yes / No
Heart conditions or circulation problems:
(angina, high blood pressure, varicose vein, aneurysm, embolism, heart attack, other)?
Do you have chest pains at any time? Yes / No
Do you suffer from fainting/blackouts/dizziness? Yes / No
Is there any history of heart disease in your family? Yes / No
7 Are you currently taking medication ? Yes / No
If yes, please give particulars:
134
8 Are you currently attending your GP for any condition or have you consulted your
doctor in the last three months? If yes, please give particulars: Yes / No
9 Have you had to consult your doctor, or had hospital treatment within the last six
months? Yes / No
10 Have you, or are you presently taking part in any other laboratory
experiment? Yes / No
PLEASE READ THE FOLLOWING CAREFULLY
Persons will be considered unfit to do the experimental exercise task if they:
have a fever, suffer from fainting spells or dizziness;
have suspended training due to a joint or muscle injury;
have a known history of medical disorders, i.e. high blood pressure, heart or
lung disease;
have had hyper/hypothermia, heat exhaustion, or any other heat or cold disorder;
have anaphylactic shock symptoms to needles, probes or other medical-type
equipment.
have chronic or acute symptoms of gastrointestinal bacterial infections (e.g.
Dysentery, Salmonella)
have a history of infectious diseases (e.g. HIV, Hepatitis B); and, if appropriate
to the study design, have a known history of rectal bleeding, anal fissures,
haemorrhoids, or any other condition of the rectum;
DECLARATION
I hereby volunteer to be a subject in experiments/investigations during the period of
20___.
My replies to the above questions are correct to the best of my belief and I understand
that they will be treated with the strictest confidence. The experimenter has explained to
my satisfaction the purpose of the experiment and possible risks involved.
I understand that I may withdraw from the experiment at any time and that I am under
no obligation to give reasons for withdrawal or to attend again for experimentation.
Furthermore, if I am a student, I am aware that taking part or not taking part in this
experiment, will neither be detrimental to, or further my position as a student.
I undertake to obey the laboratory/study regulations and the instructions of the
experimenter regarding safety, subject only to my right to withdraw declared above.
Name of subject (please print)
________________________________________________
135
Signature of Subject __________________________________ Date:
Name of Experimenter (please
print)____________________________________________
Signature of Experimenter _____________________________ Date:
136
Appendix F
Muscle Biopsy Procedure
The biopsy site should then be cleaned using an alcohol spray or wipe.
The skin, adipose tissue and skeletal muscle fascia should then be anaesthetised using
5cm3 of 1% lidocaine being injected into the biopsy site in 2.5 cm3 doses at 45°
proximal and 45° distal to the biopsy site respectively. Allow 3-5 min for anaesthetic to
take effect.
Apply chlorhexodine to skin in preparation to biopsy site and surrounding leg.
A #11 scalpel should then be used to make a 4-5mm longitudinal incision.
The “non biopsy hand” should then grip muscle on superior side of the leg. The biopsy
needle should be inserted into incision at an angle perpendicular to skin surface. Once
resistance has been met, flatten the angle of the needle to 45°. Then press the biopsy
gun button and remove needle quickly.
Allow assistant to remove tissue from needle (to be snap frozen in liquid nitrogen)
whilst applying pressure to the wound with sterile gauze.
Any subsequent passes (repeat of above) should be lateral to the previous pass.
Following the last pass, firm pressure should be applied for up to 5 mins (or until
bleeding stops).
Apply Steristrip (2 cm length) across the incision and then a dressing. Clean leg before
participant leaves the room.
Once participant leaves room, spray bed clean and wipe down.